Ribosome recycling by ribosome recycling factor (RRF) — An important but overlooked step of protein biosynthesis

Adv. Biophys.,

Vol. 32, pp. 121-201 (1996)

RIBOSOME RECYCLING BY RIBOSOME RECYCLING FACTOR (RRF) - AN IMPORTANT BUT OVERLOOKED STEP OF PROTEIN BIOSYNTHESIS

LASZLO JANOSI, HIROTO AND AKIRA KAJI

HARA,

SHIJIE

ZHANG,

Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A. Most biological scientists know protein synthesis as one of life’s vital processes which consists of three steps: peptide chain initiation, elongation, and termination. A survey of the current textbooks of biological sciences shows that practically all textbooks end the description of protein synthesis with the release of the completed polypeptide chain from peptidyl tRNA. According to these textbooks, the termination of protein synthesis begins with the placement of the termination codon at the A site of the ribosome. The ribosome then releases the completed peptide chain with the help of the termination-codon specific peptide release factors. The description of protein synthesis ends here abruptly as if the rest of the process is not dependent on a catalytic process. This is a gross oversight because there is an additional crucial step in protein synthesis; recycling of ribosomes through breakdown of the termination complex which consists of mRNA, tRNA and the ribosome. In Escherichia coli, this process is catalyzed by two factors: elongation factor G (EFG) and ribosome recycling factor (RRF, originally called ribosome releasing factor). This process clearly represents one of life’s most fundamental processes. For example, Mycoplasma genitalium, the smallest free-living organism with only about 500 genes, retains the RRF gene (I). In this review we attempt to introduce the readers to the existence 121

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of this last critical step in protein synthesis. Since the bulk of the papers published on RRF came from our laboratory, a traditional style review would be uninteresting. For this reason, this review is divided into four parts. In Section I, we summarize the characteristics of RRF. Section II describes reinitiation and translation coupling and the possible role of RRF in these processes. This is because suggestions have been made by others that ribosomes may not leave mRNA in these cases. Section III speculates on the possible role of RRF in the degradation of mRNA. Section IV deals with the possibility of eukaryotic RRF. Section I consists of data but Sections II through IV contain many speculations, hypotheses and possible experiments. This review concentrates on RRF, with which many biological scientists are unfamiliar. The main emphasis is therefore placed on the presentation of the past key experimental findings on RRF. When we discuss RRF in Sections II through IV in connection with other aspects of protein synthesis, we do not attempt to review these aspects. Instead of covering many related publications, we present examples to discuss the possible roles of RRF. Whenever possible, attempts are made to suggest experiments to prove or disprove the speculations and hypotheses. Given the importance of RRF, we feel strongly that this subject should be studied widely. If this review succeeds in arousing the interest of other laboratories in RRF, we feel that our efforts spent on writing this review are well rewarded.

I.

RIBOSOME

RECYCLING

FACTOR

(RRF)

1. Background In this subsection we briefly describe our thinking which led us to study the fate of ribosomes after they complete translation of one cistron and release the nascent peptidyl group. This study resulted in the discovery of RRF. As described below, the whole project originated from our desire to study the enzymatic release of deacylated tRNA from ribosomes. Between 1963 and 1973 our laboratory was involved in studies on the specific interaction of aminoacyl tRNA or deacylated tRNA with the complex of ribosomes and synthetic polyribonucleotides (Z-6). In 1963, prior to the establishment of the current knowledge of EFTu and EFTs (elongation factors of protein synthesis) involved in aminoacyl tRNA binding to the ribosome, we found that specific aminoacyl tRNA or deacylated tRNA binds, to our surprise, nonenzymatically to the complex of synthetic polyribonucleotide and ribosomes according

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to the sequence of the ribosome-bound polyribonucleotides (2,4). We now know, however, that nonenzymatic binding of uminoacyl tRNA is an artifact and does not occur in bacteria. However, our discovery of nonenzymatic binding of aminoacyl tRNA did have an important bearing on the biological process because this was used for the determination of the genetic code (7, 8). Nonenzymatic binding of deacylated tRNA to ribosomes, in contrast, has some physiological importance. First, under amino acid starvation, the binding of deacylated tRNA to ribosomes triggers the synthesis of ppGpp (9) which inhibits ribosomal RNA synthesis, a clever mechanism E. coli uses for stringent control of RNA synthesis (10). Second, deacylated tRNA binds to the P site of the ribosome each time an amino acid is added to the growing polypeptide. Third, deacylated tRNA is bound to the termination complex of ribosomes, which is the substrate of RRF. Since the nonenzymatic binding of deacylated tRN4 takes place according to the codon on mRNA, we reasoned that the binding of deacylated tRNA to the A site of the ribosome in C+JOwould be harmful to the natural process of protein synthesis, and therefore may be a part of nature’s control system in protein synthesis. If so, we thought, there must be a biological mechanism to remove deacylated tRNA from ribosomes. We then wondered if any factors existed which might remove the bound tRNAs from ribosomes. Our search for such factors yielded two soluble proteins we thought to be new; however, they turned out to be already known enzymes: polynucleotide phosphorylase and ribonuclease (RNase) I I (II), which digested ribosome-bound synthetic polyribonucleotides. Digestion of the ribosome-bound polyribonucleotides by these enzymes resulted in the release of the ribosome-bound tRNA. This was an interesting and surprising finding because ribosomes should protect bound RNA from these enzymes (for recent review see (12)). However, we realized that this was a mere artifact and did not represent the release of tRNA from ribosomes which may occur in bacteria. We then turned our attention to the removal of tRNA during peptide chain elongation. Using polyuridylic acid (polyU) as mRNA, we found that tRNA was indeed released by translocation (13). A significant finding in this experiment was that the ,4 site had to be occupied by either deacylated or aminoacyl tRNA for deacylated tRNA to be released from the P site by the action of EFG (13). At that time we thought that tRNA was released from the P site, because this was long before the advent of the concept of an additional site, the exit site (E

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NH2 NH2 NH2NW

plmwn pummydn plmmydn pummydn

ET AL.

RRF EFG GTP

Fig. 1. Schematic representation of the substrate for RRF. I. Puromycin treated naturally occurring polysome isolated from tetracycline treated E. coli. Top: mRNA with multiple ribosomes having growing polypeptidyl tRNA on the P site is arrested by the addition of tetracycline. Note that every ribosome has the empty Middle: The polysome depicted in the A site due to pretreatment with tetracycline. top picture is treated with puromycin and all the growing polypeptides are now converted to peptidyl puromycin. Note that deacylated tRNA which released the nascent peptide is situated at the P site of the ribosome. Bottom: The polysome without peptides depicted in the middle picture is treated with RRF, EFG, and GTP. Ribosomes and tRNA are released from mRNA. II. Naturally occurring “termination complex” at the termination codon UAG. Top: The ribosome which has just reached the termination codon. Note that peptidyl tRNA is situated on the P site and the termination codon (UAG) is on the A site. Middle: The ribosome depicted in the top picture is now treated with GTP, RF3, and RFl, which is specific for the UAG codon. The completed peptide chain is released and the “termination complex” is now formed. Note that deacylated tRNA remains on the P site and the A site is empty. Bottom: The “termination complex” depicted in the middle picture is now treated with RRF, EFG, and GTP. The ribosome and tRNA are now released from mRNA. For the sake of simplicity, the E site (25) is not shown in I or II.

site) (14, 15). Studies on RRF began with our interest in another natural occasion when tRNA leaves the ribosomes. This is after the ribosomes complete peptide synthesis and release the nascent peptide chain from the last tRNA corresponding to the carboxy-terminal of the protein. The subject of the research was the “termination complex” which con-

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BY RRF

125

sists of the ribosome, bound mRNA containing the termination codon and the P-site bound deacylated tRNA. This tRNA has just released its nascent peptide chain through the help of the peptide release factors RF1 or RF2, and RF3 (for review see (16, 17)). The structure of this “termination complex” is shown in the middle diagram of Fig. l-II. Knowing the stability of the tRNA-ribosome-polyU (mRNA) complex (we found that the complex can be isolated by the sucrose gradient centrifugation method (Z)), we postulated that, for the release of the tRNA from the termination complex, mRNA too must be removed from ribosomes either first or simultaneously with tRNA release. This hypothesis suggested that we should be studying the breakdown of the whole “termination complex.” Studies on this process led us to discover a hitherto unknown factor which catalyzes this process - RRF. We then realized that this “breakdown” must be a vital step in the protein synthesis process because living organisms must use the spent mRNA, the ribosome, and tRNA for the next round of protein synthesis. Certainly no organism can afford to dispose of its protein synthesis machinery after only one round of translation. 2. Substrate for RRF In this section, we describe the natural substrate of RRF and the model substrate we used for most of our studies. As shown in Fig. l-11, during the peptide termination reaction, the peptide release factor RF (in this case depicted as RF1 because the termination codon UAG is shown on the mRNA) together with ribosomes releases the completed chain from the tRNA corresponding to the carboxy-terminal amino acid. Since RF1 is proposed to bind at the A site (18), the resulting complex would have deacylated tRNA on the P site. RF3 is supposed to function for RF1 or RF2 (21,22) (perhaps preferentially RF2, (19)), like EFTu functions for aminoacyl tRNA (20), and removes itself from the A site (18). It is also possible that RF3, a G-protein, removes RF1 or RF2 from the A site with the use of GTP (18). At the time we started to work on the fate of the “termination complex, ” the actual complex as shown in Fig. l-11 was difficult to obtain. Therefore, we decided to use a model substrate as shown in Fig. 1-I (the middle diagram). This is a complex of mRNA with ribosomes having an empty A site and bound deacylated tRNA at the P site. As can be seen from this figure, this model complex is not identical to the true termination complex. The true termination complex has the termination codon at the A site while the model complex does not. Despite this difference, we conjectured that the metabolism of the

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model complex is similar or identical to the metabolism of the true termination complex. We postulated this because, as shown in Fig. lII, the termination codon specific events are completed before formation of the termination complex. The enzymes which catalyze the breakdown of the termination complex do not have to recognize the termination codon again. If an enzyme exists to break down the termination complex, it should function to break down similar complexes resulting from ribosomes which accidentally lose peptidyl groups or peptidyl tRNA during the peptide elongation process. For these purposes, the presumed enzymatic factor should not be specific to the termination codon. Therefore, the use of this model for studying metabolism of the termination complex appeared reasonable. How did we get the model complex pictured in Fig. l-I? Note in this figure that the model complex consists of multiple ribosomes bound to mRNA. This is because we obtained this model complex from naturally occurring polysomes. It was known that ribosomes line up on mRNA for active protein synthesis in viva. This string of ribosomes queuing on mRNA is called a polysome to distinguish it from a single ribosome (monosome) with or without mRNA. Each ribosome in a polysome should carry a nascent polypeptide chain which gets longer as the ribosomes advance toward the 3’ end of mRNA as shown in Fig. 1-I. Note in this figure that every ribosome has the A site empty, i.e., no aminoacyl tRNA is at this site. We reasoned that the string of ribosomes with no aminoacyl tRNA on the A site as shown in the top figure of Fig. 1-I can be obtained from growing E. coli treated with tetracycline immediately before polysome isolation. Tetracycline was known then to inhibit binding of aminoacyl tRNA to the A site (23). In the natural termination process, release of the nascent peptidyl group takes place by the concerted action of the termination codon specific peptide release factors RF1 or RF2, RF3, and ribosomes. Since the polysome isolated as described above does not have the termination codon at the A site, we had to release the polypeptide by other means. We used puromycin for this purpose in order to get the model substrate pictured in the middle of Fig. l-1. As shown, every ribosome of the polysome would release the nascent peptidyl group as peptidyl puromycin. This leaves the model substrate behind: a complex of ribosome, tRNA and mRNA. RRF and EFG break down this substrate into its components. As described in Subsection 8, we later used the natural termination complex as the substrate. Our substrate has labeled mRNA so that

RIBOSOME

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BY

release can be monitored

127

RRF

(see Subsection

3 for the assay).

3. Assay Method for RRF Activity With the model substrate described above, how do we measure the breakdown of the model termination complex? As pictured in Fig. l-1, the enzymatic breakdown of the model complex releases ribosomes from mRNA. This means conversion of polysomes to single ribosomes. Polysomes sediment faster than single ribosomes in sucrose density gradient centrifugation. This is easily detected by examining the sedimentation pattern of the model substrate. This system, though theoretically sound, has one serious drawback. The conversion of polysomes to single ribosomes would happen if one adds any agent which disrupts mRNA such as ribonuclease. Each single ribosome produced by ribonuclease would have short residual mRNA bound to it. This would not, however, make a great difference in the sedimentation behavior of the ribosome. Consequently, with this assay we may be isolating “new” factors which may turn out to be already known ribonucleases as we experienced when we were studying tRNAPh’ release from the polyU-ribosome-tRNAPh’ complex as described in Subsection 1. This concern accompanied us throughout the early stages of RRF research until we purified RRF to homogeneity. ,4n alternative assay method for the release of ribosomes from mRNA was devised by Tai and Davis (24). It should be noted that RRF was discovered in 1973 independently by Drs. Tai and Subramanian at Dr. B. Davis’ laboratory and identified later to be identical to ours (25,26). We describe their assay method briefly. Polysomes are isolated from [3H] uridine-pulse labeled growing E. coli. Because of pulse labeling, the label is almost exclusively on mRNA and not on ribosomes. In a similar manner to our method, the puromycin reaction is used to release the nascent polypeptide chain. The assay method, however, is different in that the RRF treated, mRNA-labeled polysomes are filtered through a Millipore filter and the ribosomebound 3H-labeled mRNA trapped on the filter is measured by counting the radioactivity on the filter. For unknown reasons, ribosomes are trapped with 100% efficiency by this filter, which has a pore size larger than the ribosome. Since ribosomes are trapped by the filter, radioactive mRNA bound to ribosomes is also trapped while released mRNA goes through the filter. This method is easier and simpler than our method described above. A third method is to use labeled R17 RNA. As described in

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ET AL.

Section I Subsection 8, the termination complex of the labeled mRNA and ribosomes is subjected to sucrose gradient centrifugation. The released radioactive mRNA in the supernatant is counted.

4. Preliminary Evidence That the Conversion of Polysomes to Single RibosomesIs Catalyzed by Factor(s) Other Than Ribonucleases or Elongation Factors. In 1970, using our assay method described above, we found that conversion of polysomes to single ribosomes was catalyzed by crude E. coli extract as shown in Fig. 2 (27). Our concern that the responsible factor(s) may be a ribonuclease was partially eliminated by three facts revealed by this experiment. First, the conversion was GTP dependent. Ribonucleases, for example RNase II, were known not to require GTP. Second, we used a polynucleotide-phosphorylase free mutant of E. coli. Third, the addition of a large amount of inorganic phosphate (a substrate of polynucleotide phosphorylase) did not increase the conversion of polysomes to single ribosomes. Next, we wished to see if already known peptide elongation factors were responsible for the conversion. As shown in Fig. 3, this possibility was eliminated by the observation that pure elongation fac-

1.5 c

E *

‘.O

c

Tbp

(A)

plysome AOtW

c

70s

Volume

1

(ml)

Bbtiom

(6)

+ soluble factor 70s

Volume

(ml)

Bl

Olll

Fig 2. Effect of various factors on the sedimentation behavior of polysome (27). Reaction mixture (A) (0.27 ml) for the breakdown of polysomes contained 10 rnM Tris-HCI (pH 7.4), 8.3 mrvt MgSO+ 50 rnM NHJZI, 6 rnM /3-mercaptoethanol, and polysome preparation from E. coli B (1.85 ODz60 units). The following were added in addition to the above components: (B) 146 1.18 of soluble proteins; (C), 0.16 IIIM GTP, 3.2 ITIM phosphoenolpyruvate, 20 /.tg of pyruvate kinase; (D) 0.16 mM GTP, 3.2 mrvr phosphoenolpyruvate, 20 /tg of pyruvate kinase, and 146 /tg of soluble proteins. The reaction mixtures were incubated at 30°C for 25 min, chilled on ice, and layered on 5 ml 15-30% sucrose gradient. After the centrifugation in a Beckman SW50.1 rotor at 38,000 ‘pm for 50 min at 4”C, absorbance at 254 nm of the gradient was plotted against the volume from the top of the tube.

RIBOSOME

RECYCLING

129

BY RRF

(4

1.5

T+G

70s 1.0 I 0.5 0 5

1.5

f fii

1.0

ih.

Aminoacyl-tRNA

0.5

TOP

Volume

(ml)

factors (EFT and EFG) and aminoacyl-tRNA on Fig. 3. Effect of two elongation the breakdown of polysome (27). Sedimentation patterns of polysomes were studied as in Fig. 2. (A) In place of crude E. coli extract, purified EFG (1.5 pg) and EFT (2Opg) were added to the reaction mixture; (B) a mixture of aminoacyltRNA (50 pg), EFG (15 /tg), and EFT (20 pg) were added to the reaction mixture instead of crude E. coli extract; (C) complete reaction mixture with crude extract of E. coli and other necessary components.

tors G and T (EFG and EFT, respectively) did not cause the conversion of polysomes to single ribosomes. A mixture of aminoacyl tRNA did not have any effect on this reaction either. Since the soluble extract was free of aminoacyl tRNA, this meant that none of the ribosomes pictured in Fig. 1-I (the middle picture) had to move along the mRNA before their release from mRNA. We believe that 70s ribosomes cannot move along mRNA without synthesizing protein. Polysome breakdown is therefore not a case of ribosome run-off. Run-off of ribosomes means that ribosomes “run” along the mRNA until either the end of the mRNA or the termination codon and jump “off’ the mRNA. Since ribosomes do not “run” in our reaction, we believe this reaction represents “release of ribosomes.” Hence, we called the factor(s) responsible for this reaction “ribosome releasing factor (RRF).” We changed the name of this factor later to “ribosome recycling factor” (28) because the ultimate function of RRF is to “recycle” ribosomes (those which have finished one round of translation) by releasing them from mRNA. Another reason for avoiding the term ribosome

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ET AL.

releasing factor was that many scientists, including those engaged in protein synthesis research, confused RRF with RFl, RF2 and RF3 (peptide release factors). We had to explain many times that RRF yeleases ribosomes from mRNA while RFs release peptide from peptidyl tRNA. By changing the name to ribosome recycling factor, we can avoid such confusion. An additional reason for calling this factor a “recycling” factor was the possibility that RRF may recycle ribosomes through dissociating ribosomes into their subunits at the termination codon in addition to the complete release of ribosomes from mRNA (see Section II and Section IV). 5. Release of Ribosomes from mRNA Requires GTP and Is Catalyzed by Two Factors: EFG and RRF Encouraged by the preliminary evidence that the release of ribosomes from mRNA was dependent on a hitherto unknown factor, we began further characterization of this factor(s) and the release reaction (29). Our first task was to determine how many factors are involved in this reaction. As shown in Fig. 4, E. coli Q13 crude extract could be fractionated by DEAE column chromatography into three fractions: fractions’ 1, 2, and 3. Fraction 1 represents those proteins which were not trapped by DEAE while fractions 2 and 3 were more acidic proteins which were trapped by DEAE. None of the fractions alone released ribosomes but the combination of fractions 1 and 3 resulted in the release of ribosomes from mRNA. We concluded that the release is dependent on at least two factors. As shown in Fig. 4, the ribosome releasing activity of fraction 3 followed closely that of EFG. This led us to suspect that the ribosome releasing activity in fraction 3 might be EFG. The fact that this activity of fraction 3 was heat stable (5 min at 6O”C, Fig. Sb) also supported this notion because EFG was known to be heat stable while most of the other soluble factors involved in protein synthesis are heat labile. Furthermore, the ribosome releasing activity of fraction 3 prepared from E. coli G-l (30) expressing temperature sensitive EFG was heat labile. Having tentatively identified fraction 3 as EFG, our attention was directed to fraction 1. Our notion that we were dealing with a hitherto unknown factor(s) was strengthened by the behavior of fraction 1 on DEAE. All the necessary soluble factors for one round of translation of mRNA without release of ribosomes were among those proteins trapped by DEAE under these experimental conditions. This characteristic of fraction 1 (which was later identified as RRF) is a very convenient characteristic because one can easily prepare an RRF-free

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BY RRF

0.7r 0.6 0.5 F w 0 0.4 1 [6 o.*0.3 -

0.1 o-

Fig, 4. Fractionation of soluble factors from E. coli extract (29). The crude E. coli proteins were eluted from DEAE Sephadex A50 with a linear gradient of pH and KCl. Fractions were collected and the absorbance at 280 nm was measured after two-fold dilution (0). Aliquots of each fraction were assayed for the factor specific polyphenylalanine activities; 0, distribution of EFT expressed as “‘C-labeled formed per 0.1 ml of the reaction mixture for the assay of EFT; *, distribution of polyphenylalanine formed per 0.1 ml. of the reacEFG expressed as “‘C-labeled tion mixture for the assay of EFG; 0, distribution of one of soluble factors (RRF) necessary for conversion of polysomes to single ribosomes. For the assay of RRF, conversion of polysomes to single ribosomes was used. The values are expressed as the percentage increase of single ribosomes in the reaction mixture.

100 0 5 80 -

(a)

Amounts of fraction 1 added (pg)

- W

Amounts of frabon

3 added (pg)

Fig. 5. Heat stability of fraction 1 and fraction 3 (29). (a) The reaction mixture for the conversion of polysomes into single ribosomes was identical to that described in Fig. 4 except that it contained, in addition to other necessary components, various amounts of fraction 1 (o), or heated fraction 1 (A); 0, fraction 1 was added but no fraction 3 was added. (b) The reaction mixture was identical to (a), except that 23 jrg of fraction 1, and various amounts of fraction 3 (0) or heated fraction 3 (A) were added; 0, fraction 3 was added but no fraction 1 was added. The amount of increase in single ribosomes during the 15-min incubation at 30°C were plotted. Heating was for 5 min at 603C.

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ET AL.

d iTP

ATP l,s&--J”Tp, 0

2

3

NTP (mM) Fig. 6. Nucleotide specificity for the conversion of polysomes into single ribosomes (29). The reaction mixture contained, in addition to necessary components, various amounts of nucleotide triphosphate, 23 pg of fraction 1 and 25.2 pg of fraction 3, and 0.08 mM puromycin. The amount of increase in single ribosomes during 15 min incubation at 30°C was expressed as the percentage of total ribosomes.

extract which catalyzes one round of translation without release of ribosomes from mRNA. Most of the characteristics of the biochemical reaction catalyzed by RRF were established at this stage. The nucleotide requirement was GTP specific, and other nucleotide triphosphates were not as efficient as GTP. Although low activity was observed with ATP and CTP (Fig. 6), this perhaps was due to phosphorylation of trace amounts of guanine nucleotide present in the system. One important feature of RRF, heat stability, was established with fraction 1 (Fig. Sa).

6.

Purijkation

of RRF to Homogeneity

By 1972 we were certain that we were dealing with a novel factor which we hoped to be important in protein synthesis. We then began a systematic purification of RRF. Due to the effort of Dr. A. Hirashima who discovered RRF (27), we obtained electrophoretically homogeneous RRF. The purification procedure consists of six steps as described in Table I (173). The major steps are DEAE cellulose chromatography (10 fold purification) and CM-Sephadex chromatography (about 7 fold purification). The DEAE step includes a heat treatment. The concentrated (5 mg/ml) RRF was heated to 60°C for 5 min. This takes advantage of RRF’s heat stability. The profile of CM-cellulose

RIBOSOME

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TABLE I Purification

of RRF

Step

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BY RRF

(173)

Fraction

--

~~~~~

Protein b-d

(x102)

7,776 3,965 210 11.2 2.47 0.21

1,165 793 618 220 75 17

__ I 2 3 4 i 6

Supernatant (NH4)ZSO+ fraction DEAE-cellulose chromatography CM-Sephadex chromatography CM-cellulose Sephadex G-SO

SF~~~~s~~~

Yield (“/)

of protein) 15 21 294 2,000 3,036 8,100

0.08

100.0 68.1 53.0 18.9 6.4 1.5

0.4

0.3

8 c .P

i

0'06

g

0.04

0.2

0.02

0.1 a z'

c fi 8

0

0 0

20

40

60

80

Fraction

Number

100

120

140

column chromatography of RRF (173). Partially purified Fig. 7. CM-cellulose RRF was subjected to fractionation on a CM-cellulose column. Absorbance at 280 nm (0) and RRF activity (0) are plotted against the fraction number. For the assay of RRF activity, 2~1 of each fraction was used in the standard reaction mixture (0.25 ml). RRF activity in the absence of EFG (A) and concentration of NH4Cl (-) are also indicated.

column chromatography of the partially purified RRF is shown in Fig. 7. An important point in this figure is that release of ribosomes by each fraction of RRF eluted from the column was completely dependent upon the addition of EFG (note A). The overall purification was 500 fold with a yield of 1.5%. The preparation was pure as examined by two dimensional gel electrophoresis (Fig. 8). With the use of E. coli overexpressing RRF (31), the purification process is much simpler because one can follow RRF protein visualized on polyacrylamide gel electrophoresis. With such overexpressed E. coli (as much as 20-30%

134

L. JANOSI Migration of Purifid

ET AL.

RR Factor on 2-D Gel RR ftom Riboromal Wash

+ H -NEPHGE

. OH-

Fig. 8. Two-dimensional gel electrophoresis of purified RRF. RRF was subjected to two-dimensional gel electrophoresis (65). The first dimension was nonequilibrium pH gradient gel electrophoresis (NEPHGE) and the second dimension was sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDSPAGE, 12.5% acrylamide). After electrophoresis, the gel was stained with Coomassie Blue.

of total protein is RRF in this cell), the purification scribed in Table I is still valid.

procedure

as de-

7. Properties of Pure RRF: Molecular Weight, Stoichiometry, and Lack of Ribonuclease Activity With the purified preparation, important information on this factor was obtained. First, we determined the molecular weight of RRF using the gel filtration technique (32). As shown in Fig. 9, the molecular weight of purified RRF was estimated at 18,000. Later, with the gel electrophoresis method we obtained the value 23,500 (33). These values are close to the value 20,639 calculated later from the deduced amino acid sequence. Second, we determined the stoichiometric relationship of RRF with EFG. As discussed above, we knew at this point that RRF’s action is dependent on EFG. However, we did not know how many EFG molecules per one RRF molecule were required for the reaction. With pure RRF and EFG available, we were able to determine a quantitative relationship between these two factors. As shown in Fig. 10, the dose response curve of EFG in the presence of a constant amount of RRF was constructed. The amount of ribosomes released from mRNA increased linearly with increasing amount of EFG up to 0.6 pg EFG in

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Pancreatic RNase A Soybean trypsin

4

1

Molecilar dight

5678

(xl 04)

Fig. 9. Molecular weight determination of RRF by Sephadex G-100 gel filtration (173). Gel filtration was carried out according to the method of Whitaker (32). KL=(V~-V,) (Vx-V,), where V, is elution volume of the sample, V, is column volume, and V, is void volume of the column, The K,,‘s of the samples were plotted against the log of the molecular weight of the proteins used as standard. (----) K,,,, of RRF and the corresponding molecular weight of RRF.

the reaction, but then RRF became saturated. From the molecular weight of RRF (18,000) and that of EFG (84,000) we could estimate that approximately 12.8 picomoles of EFG saturated 10 picomoles of RRF. This calculation suggests that the stoichiometric relationship between RRF and EFG is 1 :l. This finding is consistent with the notion that the release of ribosomes is achieved by the concerted action of each molecule of RRF with another molecule of EFG. Further evidence in support of this notion will be presented later in Subsections 11 and 17. Third, we wished to prove that RRF is not a ribonuclease which may break down mRNA. For this purpose, we incubated isolated [‘“Cl uracil-labeled mRNA with pure RRF and other components of our assay system in the experiment shown in Fig. 11. The incubation did not change the sedimentation profile of the labeled RNA at all. This shows that no ribonuclease-like activity was present in pure RRF. We then wished to establish that the mRNA of the polysome from which ribosomes are released by RRF is not degraded during the release of ribosomes. In the experiment shown in Fig. 12, polysomes

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04-H-H+-

0.3 0.6 0.9 1.2 IS

ET AL.

I.6

~9 of EFG oddcd Fig. 10. Stoichiometry between RRF and EFG for the release of ribosomes from mRNA (173). The reaction mixture (0.25 ml) for the release of ribosomes from mRNA contained polysomes, other necessary components, 0.18 pug of purified RRF and various amounts of purified EFG. The reaction mixture was incubated for 15 min at 3O”C, and the sedimentation behavior of ribosomes was analyzed on a 1 S-30% sucrose gradient.

lob

10

20

Tub6 Number Fig. 11. Lack of ribonuclease activity of RRF and EFG in the presence of polysomes (173). The complete reaction mixture (0.25 ml) for the assay of ribonuclease activity of RRF and EFG was identical with that for the assay of RRF activity. It contained, in addition to other necessary components, 1.9 ODZ~ units of unlabeled polysomes, 0.23 pg of purified RRF, 1 pg of purified EFG, and 8,000 cpm of [“Cl uracil-labeled mRNA. The reaction mixture was incubated at 30°C for 15 min. RNA was isolated by the phenol extraction method. The labeled RNA was fractionated on a linear sucrose density gradient (S-20%). The trichloroacetic acidinsoluble radioactivity of the fractions was measured and plotted against the tube number. (A) Unlabeled polysomes, RRF, and EFG were omitted from the complete system; (B) unlabeled polysomes were omitted; (C) RRF and EFG were omitted; (D) complete reaction mixture. Sedimentation was from left to right.

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(A)

2M)

TOD

1

2

I 3

Volume (ml)

1 I 4

I I 5

Top

10

20

Tube Number

Fig. 12. Evidence for preservation of mRN4 during the release of ribosomes from mRNA by RRF and EFG (173). For the conversion of labeled polysomes into single ribosomes, the reaction mixture (0.5 ml) was identical with the standard complete reaction mixture except that it contained 3.9 OD260 units of [“Cl uracil pulse-labeled polysomes (containing 16,000 cpm of mRNA). The reaction mixture was incubated at 30°C for 15 min and divided into two parts at the end of the reaction. One part was used to detect the conversion of polysomes into single ribosomes, and the other for the extraction of mRNA from the polysomes. (1) RRF and EFG were omitted from the complete reaction mixture; (2) complete reaction mixture; (3) 3.25 OD16” units of rRNA were added to the complete reaction mixture. (A) Sedimentation behavior of ribosomes after the treatment; (B) sedimentation behavior of mRSA isolated from labeled polysomes used in (A).

with pulse-labeled mRNA were used as substrate for RRF and EFG for the release of ribosomes. The sedimentation profile of the pulselabeled mRNA isolated from the reaction mixture was almost identical to the intact mRNA (compare Fig. 11 (A) with Fig. 12 B (3)). It should be noted that almost complete release of ribosomes from mRNA took place (compare Fig. 12 A (1) with Fig. 12 A (3)) while the mRNA remained intact (Fig. 12 B (3)). Because of the presence of a trace amount of ribonuclease in the polysome preparation, the mRNA isolated from the polysome was slightly digested (compare Fig. 12 B (1) with Fig. 11 (A)). However, the important point is that the release of the ribosome from the mRNA by RRF and EFG did not change the size of this mRNA. To prevent the action of polysomal ribonuclease on mRNA, an excess of ribosomal

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ET AL.

RNA was added to the reaction mixture as indicated in Fig. 12 A (3) and B (3). It prevented the slight degradation of mRNA but did not inhibit release of ribosomes from mRNA by RRF and EFG. We concluded that RRF is not ribonuclease. 8. RRF Releases Ribosomes from mRNA at the Naturally Occurring Termination Codon Up to this point, our studies were performed with the model substrate: puromycin-treated polysomes. We still had to demonstrate that the major function of RRF is to recycle ribosomes through the release of ribosomes from mRNA at the termination codon. From 1975 until the molecular biological gene technology became available, our major ef-

Sequence of amB2 R17 RNA (the coat cistron Fig. 13. polymerase cistron are shown) (46, 184, 185).

TABLE II Effect of the Removal Peptide from Ribosome

of Asparagine (37)

on Release

and the beginning

of Ribosome-bound

amB2

Ribosomebound mRNA

Components

mRNA

R17

of the

RNA

and

released

pm01 Complete system + asparaginase, + asparaginase, - RRF

- Asn, - RRF - Asn

0.72 2.18 1.64 2.11

1.46 0 0.54 0.07

The complete system (0.5 ml) contained the initiation complex of ribosomes and labeled amB2 R17 RNA, EFG (13 pg), EFT (mixture of EFTu and EFTs, 52 yg), mixture of tRNA from E. coli (50 pg), crude peptide release factor RF1 (144 pg), ATP (0.4 mM), phosphoenolpyruvate (alanine, serine, phenyl(4 mM), GTP (0.2 mrvt), pyruvate kinase (15 pg), amino acid mixture alanine, threonine, and asparagine), and RRF (0.6 pg). The mixtures were incubated at 33’C for 15 min and analyzed on sucrose gradients. Where indicated, asparaginase (0.4 pg) was added.

RIBOSOME

RECYCLING

BY RRF

139

forts in this project were concentrated on demonstrating RRF function on a naturally occurring, defined mRNA with the termination codon at a defined position. These studies were important because there was a suggestion that the mechanism of ribosome release from mRNA at the termination codons may differ from the mechanism of ribosome release from puromycin-treated polysomes (34). To study ribosomal release at termination codons, we chose R17 amB2 phage RNA (35, 36). The sequence of the coat cistron together with the intercistronic sequence and the beginning of the 3’ distal cistron, the polymerase cistron, is shown in Fig. 13. The amB2 phage has UAG at the seventh codon of the coat cistron. The 4th amino acid in this cistron is ,4sn and the 8th, 9th, lOth, and 1 lth amino acids are Phe, Val, Leu, and Val, respectively. These amino acids are important in understanding the experiments described in this and the next sections. In the experiment described in Table II (37), amB2 phage RNA was labeled with 3H and ribosomes were bound to the initiation sites of this RNA. The major in oitro protein synthesized under the direction of R17 RNA is the coat protein. Therefore, the majority of the initiation complex is that of the coat cistron. This complex was isolated to free the complex from the initiation factors used to make the complex. To this complex, purified components necessary to bring ribosomes to the sixth codon, including Ala, Ser, Asn, Phe, Thr, and peptidyl release factor 1 (RFl) were added. RF1 is C,4G specific and releases hexapeptide corresponding to the -NH?-terminal six amino acids of R17 coat protein from peptidyl tRNA. Then, release of ribosomes from the labeled R17 RNA by RRF was monitored by sucrose gradient centrifugation. As shown in Table II, when RRF was omitted, release of R17 RNA was reduced to l/20 of the complete system indicating that RRF indeed releases ribosomes from natural mRNA at the natural termination codon. The fact that the ribosome has to be at the termination codon to be released was demonstrated by the observation that removal of Asn from the system also significantly reduced the release of R17 RNA by RRF. In the above experiment, chain elongation with six amino acids was allowed in the reaction mixture starting with the isolated initiation complex of the coat cistron. In other words, the termination complex was mixed with the elongation components of protein synthesis. We then asked the question whether or not RRF can work on the isolated, purified termination complex of tRNA and the ribosome having the R17 coat mRNA’s 7th CAG codon at the A site. To answer this question, instead of starting with the initiation complex, ribosomes

140

L. JANOSI

TABLE III Requirement of RRF for Release somes at the Termination Codon

of mRNA (37)

from

the Complex

of tRNA,

mRNA,

Ribosome-bound

ET AL.

and Ribo-

Released

Components [‘HI Complete - GTP - RRF - RRF,

system

mRNA

(pmol)

0.96 1.72 1.52 1.65

+ PM

0.76 0 0.20 0.09

The complete system (0.25 ml) contained the termination ribosome complex, 8 mM Tris-HCl 40 mM NH,Cl, 3.2 mM phosphoenolpyruvate, 0.16 mM GTP, 7.8 (pH 7.4), 8 mM Mg(OAc)l, pg pyruvate kinase, EFG (2.4 pg), RRF (0.43 ,ug), and ribosomal RNA (0.1 mg). Puromycin (PM) (50 PM) was added where indicated. After incubation at 37°C for 15 min, the mixtures were analyzed on sucrose gradients. TABLE Release

IV of mRNA

from

the Termination

Complex

in the Presence

of Puromycin

Ribosome-bound

mRNA released

Components mRNA

(37)

Peptides pm01

Hexapeptidyl-tRNA +PM +PM, +EFG +PM, +EFG, +EFG, +RRF

complex

+RRF

0.26 0.26 0.20 0.00 0.20

0.17 0 0 0 0.11

0 0 0.06 0.26 0.06

reaction mixture (0.25 ml) contained hexapeptidyl complex, 50 pM PM, 13 yg EFG, 0.2 GTP, and 0.43 pg RRF where indicated. The mixture was incubated at 37°C for 15 min, and 0.22 ml of the mixture was analyzed on a sucrose gradient. The

mM

were brought to the 6th codon and the peptide was released by RFl. The complex was then freed of GTP and other protein synthesis soluble factors. Table III (37) shows that release of ribosomes from amB2 RNA at the termination codon was dependent on RRF. It is important that the GTP requirement was clearly demonstrated in this experiment. Since ribosomes had already moved to the 6th codon and released hexapeptide by the action of RFl, there was no need for GTP except in the release of ribosomes from amB2 R17 RNA. Removal of GTP from the system completely stopped the release of ribosomes from amB2 RNA. The addition of puromycin to the system with RF1 which had already released the hexapeptide did not permit the release of ribosomes from amB2 RNA if RRF was omitted.

RIBOSOME

RECYCLING

BY RRF

141

To demonstrate that the model system is valid, we used puromytin instead of RF1 in the next experiment. As described previously, in early studies, we used our model system which consisted of polysomes treated with puromycin. In the experiment described in Table IV (37), the termination complex of hexapeptidyl tRNA, ribosome, and [3H] amB2 RNA was isolated. Instead of RFl, puromycin was added to release the peptide of peptidyl tRNA. All peptidyl groups were released from the termination complex by puromycin. In contrast, ribosomes were released from [3H]-labeled amB2 RNA only in the presence of both RRF and EFG. The requirement for EFG was clearly demonstrated. A very important fact shown in Table IV is the puromycin requirement for the release of ribosomes from mRNA. RRF should not release ribosomes from mRNA when ribosomes are engaged in chain elongation. What prevents RRF from releasing ribosomes from mRNA during the chain elongation process? It is the ribosome-bound peptidyl tRNA. For the release of ribosomes from mRNA, the peptidyl group bus to be released first either by puromycin or the peptide chain release factor (RFl, RF2, and RF3). The puromycin requirement shown in the model system was therefore confirmed using the natural substrate. In summary, we believe that the model substrate we used to study the fate of the termination complex was legitimate. All information which we obtained with the model system was confirmed using the termination complex of amB2 RNA. 9. Serious Consequences of the Absence of RRF in In Vitro Protein Synthesis What would happen if RRF were absent from the protein synthesizing system? We can imagine the following possibilities: 1) Ribosomes “stop” at the termination codon, simply stay there, and do nothing. 2) Ribosomes release their SOS subunits but 30s subunits simply stay there until they accidentally fall off by mechanical or thermal disturbance. 3) Ribosomes release their SOS subunits, and 30s subunits move toward the 3’ end of mRNA or the remaining 30s subunits rapidly scan the mRNA until they come to a place to stop, such as the initiation codon with the Shine-Dalgarno sequence. 4) Ribosomes “stop” and release the peptidyl group with the help of peptide release factors (RFs), and pick up aminoacyl tRNA corresponding to the 3’ distal codon next to the termination codon. They then continue to move toward the 3’ end and synthesize polypeptide coded for by the 3’

142

L. JANOSI

TABLE V Suppression

of the Amber

Mutation

by Suppressor

tRNA

[$H] valine

Is Dependent

incorporation

(cpm)

Control

+ RRF

on RRF

ET AL.

(44)

tRNA Control Wild type Suppressor

540 538

Suppression 2

117 224

Suppression

107

The reaction mixture (0.06 ml) contained 18 pmol of well-washed ribosomes containing the initiation complex with amB2 R17 RNA and fMet-tRNA (18 pmol of ribosomes), 86 /rg of E. coli crude extract free of RRF, mixture of amino acids (- Val) (5 pmol each), 1 /Ki of [3H] valine (1 mCi/0.0464 mg), 0.14 rnM GTP, 1 rnM ATP, 2.7 rnM phosphoenolpyruvate, 7.8 /rLg of pyruvate kinase, 10 rnM Tris-HCl (pH 7.4), 200 rnM NH4C1, 10 rnzvt MgSO+ and 45 pug of suppressor tRNA or 100 ,ng of wild-type tRNA. Where indicated, 0.43 pg of RRF was used. After incubation at 37°C for 15 min, the hot trichloroacetic acid (So/,) insoluble radioactivity was counted. Suppression was calculated by subtracting the cpm value obtained with the wild type from that obtained with the suppressor tRNA.

distal sequence of mRNA. 5) The 70.5 ribosome stays on mRNA and starts moving toward the 3’ end of mRNA without peptide synthesis. This would be the so-called “phaseless” and “sterile” travel (38-42). With the use of amB2 R17 phage RNA as described in the preceding section, we determined which of the above possibilities was correct. Since there is an amber mutation at the 7th codon of the coat protein, the amino acid incorporation into proteins directed by amB2 RNA should be much less than that directed by the wild type R17 RNA. The hexapeptide corresponding to the NH2 terminal of the amB2 coat protein is acid soluble; therefore, production of the aminoterminal hexapeptide under the direction of amB2 R17 RNA would escape the detection by radioactive amino acid incorporation into acid insoluble fractions. The amino acid incorporation into coat protein should, however, increase in the presence of the amber suppressor tRNA which reads the amber codon as Ser (43). This is what was observed and shown in Table V (44). An important observation, however, is indicated in this table under the heading of “control”. In this control, the extract was devoid of RRF. Incorporation of Val into the coat protein was similar whether or not the amber suppressor tRNA was present. In other words, the absence of RRF increased amino acid incorporation into acid insoluble proteins, and the addition of amber suppressor tRNA did not increase this protein synthesis further. These data support possibility 4 from above, i.e., the ribosomes which are not released by RRF may continue to translate the mRNA.

RIBOSOME

RECYCLING

BY RRF

TABLE VI Quantitative

Relationships

of Various

amB2

R17 RNA

143 Coat

Polypeptides

Synthesized

under

the Direction

of

(45) Product

Peptide Without Cf (formylmethionyl coat) Cm (methionyl coat) Cd (coat protein lacking NH>-terminal NH>-terminal hexapeptide

7AA)

5.3 1.2 30.1 51.6

RRF

(pmol

x 10’) ~-.-

~--.

With

RRF

4.0 1.1 8.4 165.0

The content of formylmethionine was determined by analysis of pronase digests of [j’s] methionine-labeled protein synthesized under the direction of R17 amB2 RNA. The amount of X+-terminal unblocked methionine was measured by the first cycle of Edman degradation. The quantities of Cf, Cm, and Cd presented in this table correspond to the product synthesized from 40 pg of amB2 R17 RN.4 in the absence or presence (1.7 pg) of purified RRF.

Based on these preliminary results, we carried out more detailed studies on the consequence of removal of RRF from this system. First, we analyzed and quantified all polypeptides and oligopeptides produced in the presence of RRF. We found that in the complete in vitro protein synthesis system, essentially four kinds of products were made under the direction of amB2 R17 RNA. They were the coat protein with formylmethionyl group at the NH:! terminal, (Cf) coat protein with methionine at the NH2 terminal (Cm), the coat protein without the seven NHz-terminal amino acids (Cd) and the NH*-terminal hexapeptides (fMet * Ala * Ser * Asn * Phe * Thr) corresponding to six codons of the NH*-terminal sequence of the coat protein. The quantities of these products are listed in Table VI (45) (see the column titled “with RRF”). We also measured the quantity of these peptides in the absence of RRF. Table VI shows that approximately 4 times more Cd was synthesized in the absence of RRF than in its presence. In the presence of RRF, most of the product was the NH*-terminal hexapeptide. Very small amounts of coat protein, either Cf, Cm, or Cd were synthesized. This indicated that the amber codon in the 7th codon was functioning well and that over 90% of the ribosomes which had advanced up to the 6th codon terminated peptide synthesis because of the amber codon at the 7th. In the absence of RRF, a significant change was seen. At the expense of the NHz-terminal hexapeptide, a large increase of Cd was observed. The amount of Cd was not greatly different from that of the

144

L. JAN09 Al me EC-3cda cntmn a

ET AL. 3

2

RR 5

UAG-

/

EFG GTP

Fig. 14. Schematic representation of what goes on at the termination codon in the presence and absence of RRF (186). RR= ribosome recycling; EFG = elongation factor G; T. F. = termination factor (peptide release factor);@. l designates p o 1ypeptldes remmated from the codon next to the termination codon; m designates peptide released at the termination codon UAG.

NHZ-terminal hexapeptide. This suggested that most (60%) of the ribosomes which had synthesized the NH*-terminal hexapeptide might have continued to synthesize Cd. The decrease of the NH*-terminal hexapeptide in the absence of RRF was understandable because ribosomes in the absence of RRF would be busy translating the distal portion of the coat cistron and fewer would be available for synthesis of the NH*-terminal hexapeptide. Very important information was obtained when we analyzed the NHz-terminal amino acid sequence of Cd, The polypeptide Cd containing 14C-labeled amino acid was purified by polyacrylamide gel electrophoresis and subjected to Edman degradation analysis as well as fluorodinitrobenzene NH*-terminal sequence analysis. The amino-terminal peptide sequence analysis showed Phe* Val * Leu * Val, corresponding exactly to the 8th through 1 lth residues of the R17 coat protein (see Fig. 13) (46). A schematic representation of protein synthesis in the presence and absence of RRF based on these experimental results is shown in Fig. 14. The peptide with the black balls corresponds to Cd in this experiment. In the absence of RRF, the ribosomes which have released the NHz-terminal hexapeptide at the amber codon keep translating the 3’ region of the RNA from the codon next to the amber codon. We believe that the translation of the 3’ region of the coat protein must be carried out by the same ribosomes which completed the syn-

RIBOSOME

RECYCLING

BY RRF

145

thesis of the NHZ-terminal hexapeptide, since the amount of the hexapeptide was similar to that of Cd within experimental error. Furthermore, if the synthesis of Cd had been carried out by other ribosomes, they would have had to use the regular mechanism of initiation, i.e., involvement of formylmethionyl tRNA. The NH*-terminal analysis of Cd clearly indicated that no such amino terminal existed in Cd. In addition, no Shine-Dalgarno sequence is present near the seventh amber codon of amB2 R17 RNA to guide the new 30s subunits to initiate at this point. Thus, among the five possibilities for protein synthesis in the absence of RRF listed in the beginning of this section, possibility 4 is most likely. We have not yet ruled out the other possibilities completely, but have proven that most of the time case 4 occurs. This finding, as supported later by in aivo experiments, has profound biological significance. It shows that the termination complex has to be broken down by RRF and EFG; without breakdown, the ribosomes will unnecessarily translate the 3’ region of mRNA. In addition, the translation of the 3’ distal cistron (polymerase) may be hampered by the flow of ribosomes translating the distal cistron in an out of frame manner. 10. Dependence of Protein Synthesis on RRF As described in the preceding subsections, RRF was discovered strictly through its biochemical action, i.e., the release of ribosomes from mRNA. It is therefore natural for us to assume that this is an essential factor for protein synthesis. However, this has to be proven unequivocally, especially in view of the recent finding that a protein whose sequence is extremely similar (up to 65%) to that of RRF may be located in the nuclei of plants (discussed in Section I Subsection 17). Since no protein synthesis occurs in the nucleus, this finding suggests that the “plant RRF” may have functions other than protein synthesis. We present in this section the evidence that RRF is indeed a factor involved in protein biosynthesis in bacteria. For this purpose, we used two systems: de nova protein synthesis and amino acid incorporation into protein (a conventional way of studying protein synthesis). In the experiment described in Fig. 15, T4 phage lysozyme was synthesized in aitro under the direction of T4 phage mRNA isolated from T4 phage infected E. coli. The amount of T4 lysozyme synthesized as measured by its enzymatic activity was dependent on the amount of ribosomes until the system was saturated with ribosomes. The stimulatory effect of RRF was clearly observed (up to 7-8 fold) when the ribosome concentration was limiting. Even in

146

L. JANOSI

Ribosomes

[OD,,,

ET AL.

umt]

Fig. 15. Stimulation of T4 lysozyme synthesis in vitro by RRF (33). T4 lysozyme was synthesized in vitro under the direction of T4 late mRNA in the presence of various amounts of ribosomes. Where indicated, 0.6 pug of RRF purified from SlOO was added to the reaction mixture (60 ~1). The data shown were obtained from the standard lysozyme assay method (187). The bar indicates the range of deviation.

A

MW

66k

43k

25Sk

14k

BPB

MW

66k

43k

253

14k

BPB

Fig. 16. Stimulatory effect of RRF on the synthesis of various proteins programmed by T4 phage mRNA (33). T4 late mRNA was translated in the presence or absence of RRF in a reaction mixture containing 45 &i of [%!I] methionine. Purified RRF (3.4 pg) from SlOO was added where indicated. A 5 jd aliquot of the 70 ~1 reaction mixture was analyzed by SDS-PAGE, and fluorography was performed. The pictures were obtained after 7 hr (picture A) and 16 hr (picture B) exposure to X-ray films at - 80°C. The densitometric traces of these autoradiograms are shown. -, in the presence of RRF; .,.,..,.,., in the absence of RRF.

RIBOSOME

RECYCLING

BY RRF

147

the presence of near saturation concentration of ribosomes, RRF stimulated de no00 synthesis of T4 lysozyme approximately two fold. We then investigated the effect of RRF on protein synthesis as measured by the incorporation of radioactive amino acids into proteins. This was also carried out under the direction of T4 phage mRNA. Since the mRNA used was isolated from T4 phage infected E. co& this mRNA preparation also contained mRNAs for many T4-related proteins other than the one for T4 lysozyme. The radioactive proteins thus synthesized in vitro were a mixture of T4-phage related proteins. They were fractionated on SDS-PAGE, and radioactivities of protein bands were counted and plotted as shown in Fig. 16. The amount of radioactivity incorporated into every fraction in the presence of RRF (solid line) was higher than those in the absence of RRF (broken line). In support of the above findings, independent work by Kung et al. (26) showed that RRF is essential for in vitro synthesis of pgalactosidase. In 1977 Kung et al. were interested in defining soluble protein factors necessary for de nova protein synthesis. In the system where transcription and translation were coupled, they were able to observe a net increase of /3-galactosidase. Using fractions partially devoid of protein factors, a purified fraction from the “ribosome wash” was identified as a required factor for the DNA-dependent in vitro synthesis of P-galactosidase. This factor, previously called La, was purified to near homogeneity and had a molecular weight similar to that of RRF. The purification procedure was similar to that of RRF

(26). The important fact about this experiment is that La was purified according to its stimulatory effect on de no~o synthesis of P-galactosidase in vitro. This pure protein had the RRF activity. Further evidence that RRF is identical to La was provided by immunological cross reaction using the Ouchterlony technique. The stimulatory effect of RRF on P-galactosidase synthesis was approximately four fold. Based on our observations and those of Kung et al., we conclude that RRF is a factor for protein synthesis. This conclusion is supported by the presence of an elongation factor signature (except for one amino acid) in the amino acid sequence of a Haemophilus injk-nzae protein which has 69% direct identity with E. coli RRF (discussed in Section I Subsection 17). 11. Mechanism of Action of RRF: The Role of Mg2’, EFG, and GTP It is common knowledge among scientists in this field that in vitro release of ribosomes from mRNA can easily be observed if the Mg?-+

148

L. JANOSI

ET AL.

concentration is lowered below 3.5 mM (29). It is also known that the association of synthetic polyribonucleotides with ribosomes is dependent on Mg” concentration (47). It was therefore very important to examine the optimum concentration of Mg2+ for RRF. As shown in Fig. 17, RRF appears to function well at 5 mM Mg*+ which is within the physiological range of the Mg2+ concentration in viva (48). In this experiment, a model substrate, puromycin treated polysome, was used and the formation of 70s ribosomes was followed. It should be noted that most early studies on in vitro protein synthesis with synthetic polyribonucleotides utilized relatively high levels of Mg2+ in which RRF is not functional. This means that most of these early studies involved only one round of translation. As pointed out earlier, in the presence of a large amount of ribosomes, one would observe a reasonable amount of amino acid incorporation into proteins with one round of translation. This may explain why RRF escaped the attention of researchers. Because the reaction we are studying is dependent on two factors, EFG and RRF, one wonders if they work stepwise or simultaneously.

100

80

60

I

t

Ol

5

10

Concentration

of MgS04

15 (mid)

Fig. 17. Effect of MgZ+ on release of ribosomes from mRNA (174). The reaction mixture (1.25 ml) containing 11.3 A260 units of polysomes, 0.16 mM GTP, 0.84 mM puromycin, 2.95 pug RRF, 7 pug of EFG, other essential components and various concentrations of MgSO+ was incubated for 15 min at 30°C. Aliquots (0.21 ml) were analyzed for sedimentation behavior of polysomes. The amounts of 70s ribosomes (percentage of total ribosomes) present at the end of the incubation period were plotted against the concentration of MgS04 in the reaction mixture. A complete system with RRF, EFG, GTP, and puromycin; X, RRF was omitted; 0, RRF and EFG were omitted; 0, EFG was omitted.

RIBOSOME

RECYCLING

BY RRF

149

Fig. 18. Possible mechanisms of RRF and EFG action. (I), The ribosome reaching the termination codon with nascent peptidyl tRNA. The broken line is peptidyl group and the pin-like figure is tRNA. The horizontal line is mRNA. The termination codon is represented by UAG; (II) complex of release factors 1, 3, and GTP with (I); (III) the ribosome just released the completed peptide chain through collaborative action of RFl, RF3 and ribosomal RNA; (IV) termination complex which was cleared of RF3 and RF1 with the use of GTP energy. A) stepwise action in which RRF may act first; B) stepwise action in which EFG may act first; C) simultaneous action of EFG and RRF: In A, B, and C, action of EFG is depicted as causing weakened subunits association where ribosomal subunits are obliquely placed. Association of factors as depicted is purely hypothetical.

Three possible mechanisms of RRF action in conjunction with GTP and EFG are schematically represented in Fig. 18. Figures (I) to (I I I) represent an accepted scheme for the termination step. Although not well established, the reaction from complex III to IV, the removal of peptide release factors catalyzed by GTP and RF3, is also shown. This is likely to be true especially in view of the recent reports suggesting that RF3 is a G-protein (21, 22). The mechanisms A, B, and C are theoretically possible modes of action for RRF and EFG. Mechanism A postulates that RRF binds to the A site of the

150

L. JANOSI

ET AL.

termination complex. This bound RRF pushes off the P-site bound deacylated tRNA with the help of EFG. The release of tRNA should go through the third ribosomal site called the exit site (E site, (15)). For simplicity, the E site is not shown in this picture. For this movement, GTP is necessary. After this translocation-like movement, RRF releases the ribosome from mRNA with simultaneous removal of itself from the ribosome. Mechanism B starts from complex III. Thus, EFG uses RF1 which is at the A site of the ribosome for pushing off the tRNA at the P site (13). In mechanism C, the reaction starts with complex IV. EFG and RRF work simultaneously to break down the termination complex (IV). Although this scheme shows these two factors bound to the A site, we have no evidence for or against this point. To accommodate the data which indicate that freshly released ribosomes are prone to dissociate into their subunits, the released ribosomes are pictured as obliquely placed subunits (see Subsection 13). We present evidence which suggests that mechanism C is likely. In the experiment described in Table VII, polysomes, puromycin, EFG, and GTP were incubated for 10 min. RRF was then added, and incubation was continued. During the first 10 min of incubation, fusidic acid (an inhibitor of EFG) was added at time 0, 5 min or 10 min. Even if the addition of fusidic acid was at 10 or 5 min after onset TABLE Evidence Together Experiment 1 2 3 4

VII That RRF Can with RRF (I 74)

Release

Ribosomes

Components

from

Polysomes, Polysomes,

PM, PM,

EFG, EFG,

GTP GTP,

Polysomes, Polysomes,

FA PM, PM,

EFG, EFG,

GTP GTP

Only

added At 5 min

At 0 time

mRNA

FA

When

EFG

At 10 mjn

Increase of 70s ribosome (% total ribosomes)

RRF RRF

37.6 0

RRF RRF,

FA

0.6 1.2

Functions

Inhibition (%I

100 98.4 96.8

The reaction mixture (0.25 ml) for the release of ribosomes from mRNA contained 2.1 A~60 units of polysomes, 9.7 ,ug of RRF, and 19 pg of EFG and other components for conversion of polysomes to single ribosomes. Where indicated, fusidic acid was added at various times after the onset of the reaction. In Experiment 4, RRF and fusidic acid were added at 10 min after the onset of the incubation. All reaction mixtures were incubated for an additional 15 min at 30°C, making the total incubation period 25 min. The mixtures were then analyzed on linear sucrose gradients (1 S-30%). The amounts of 70s ribosomes after the incubation are expressed as the percentage of total ribosomes. FA, fusidic acid.

RIBOSOME

RECYCLING

151

BY RRF

0.1 0 0.4 E f

0.3

;

0.2

Q 5 CJ

0.1

g 0

0.4

0

0.3 0.2 0.1 0 Top

Volume

(ml)

of polysomes with EFG does not eliminate requirement of Fig. 19. Pretreatment EFG for release of ribosomes from mRNA (17-1). The pretreatment reaction mixture (1.1 ml) contained 14.8 A260 units of polysomes, 0.11 m&t puromycin, 57 pg of EFG, and 0.18 mM GTP and other necessary components. The reaction mixture was incubated at 30°C for 20 min and isolated as the pretreated polysomes (free of EFG). (A) The reaction mixture (0.25 ml) for release of ribosomes from the pretreated polysomes did not contain GTP, puromycin, RRF, or EFG; (B) 3 pg of RRF and 0.16 rnM GTP were added to (A); (C) 11.4 fig of EFG were added to (B). In this experiment, none of the reaction mixtures (A, B, and C) contained puromytin. After incubation for 15 min at 3O”C, the reaction mixtures were subjected to sucrose density gradient centrifugation for analysis of polysomes.

of the reaction, release of ribosomes from mRNA was completely inhibited. This means that the action of EFG during the 10 min preincubation period did not “prepare” the substrate for the subsequent action of RRF. This suggests that EFG alone could not function for the partial reaction of release of ribosomes from mRNA. This experiment eliminated mechanism B. The problem in this experiment is the fact that fusidic acid “freezes” EFG on the ribosome (49). The presence of fusidic acid and EFG on the ribosome may interfere with the subsequent action of RRF, even though preincubation with EFG successfully prepared the substrate for the action of RRF. Our conclusion from the above experiment, despite the problem stated above, was further supported by the experiment shown in Fig. 19. In this experiment, polysomes were pretreated with EFG and pu-

152

L. JAN03

0.4

-

TOP

ET AL.

(4

Volume

(ml)

Pretreatment of polysomes with RRF does not eliminate requirement of Fig. 20. RRF for release of ribosomes from mRNA (174). The reaction mixture for pretreatment was identical to that described in Fig. 19, except that it contained 16.4 Azao units of polysomes and 15 pug of RRF instead of EFG. The pretreated polysomes were isolated free of RRF. The reaction mixture for the release of ribosomes from pretreated polysomes contained 0.54 Arm units of the pretreated polysomes. (A) Pretreated polysomes alone were incubated; (B) 3 pg of EFG and 0.16 mM GTP were added to (A); (C) 11.4 @g of RRF were added to (B).

romycin and subsequently purified free of EFG. RRF was then added to these isolated polysomes. Although there was some conversion of polysomes to single ribosomes, release of ribosomes from mRNA was significantly diminished when compared to the reaction where RRF and EFG were added simultaneously. This experiment again rules out mechanism B. In the experiment described in Fig. 20, the polysomes were treated initially with RRF followed by isolation of this pretreated complex to free it from RRF. The purified pretreated polysomes were then mixed with EFG and the conversion of the polysome to single ribosomes was examined. Very few single ribosomes were formed under these conditions. This result ruled out mechanism A because the pretreatment with RRF did not eliminate the RRF requirement. We believe that these results leave (C) as a viable possibility (Fig. 18). This mechanism is also consistent with the presence of the Gprotein-reacting amino acid sequence motif in RRF as described in

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Section I Subsection 17, and the 1:l stoichiometric RRF and EFG (Section I Subsection 7).

relationship

of

12. Release of the Ribosome-bound tRNA by RRF and the Biological Activity of Released Ribosomes In Subsection 1 we indicated that studies on RRF were initiated from our desire to elucidate the release of the ribosome-bound deacylated tRNA from the ribosome. We present in this section evidence that the ribosome bound tRNA of the termination complex is indeed released by RRF and EFG. In the experiment shown in Table VIII, attention was focused on the fate of tRNA during the release of ribosomes from mRNA. The amount of the bound tRNA was measured by the amino acid accepting capacity. All of the bound tRNA was released during the release of ribosomes from mRNA. The maximum release of tRN,4 was observed in the complete system and omission of one or more of the components decreased the release of tRNA. If RRF functions to “recycle ribosomes,” the ribosomes released by RRF must be biologically active. This point is addressed in the experiment described in Fig. 21. This experiment was important because there was a possibility that RRF may be releasing ribosomes from mRNA by virtue of some degradative activity (for example, proteolytic activity). In this experiment, released ribosomes were collected and incorporation of radioactive amino acids into proteins by these ribosomes was examined in the presence of MS2 phage RNA. As controls, 70s ribosomes were prepared from the same polysomes without

TABLE Release

\‘I11 of tRNA

from

Reaction

Polysomes Polysomes, Polysomes, Polysomos, Polysomes, Polysomes, Total tRNA

Ribosomes

during

mixture

Release

of Ribosomes

from

mRNA

(174

tRNA released (expressed as [‘“Cl aminoacyl-tRNA) (cpm)

alone PM PM, RRF PM, EFG PM, EFG, GTP PM, RRF, EFG, GTP bound to polysomes

The amounts of tRNA released from 2.08 A Zh,, units of polysomes capacity to accept [“Cl amino acids. The formation of [‘*Cl aminoacyl radioactivity insoluble in cold (0°C) trichloroacetic acid.

222 423 239 371 642 1,048 953 are expressed as their tRNA is shown as the

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pg of ribosomes odded Fig. 21. Biological activity of released ribosomes (174). Released ribosomes were prepared from E. coli Q13, and ribosomal activity was measured by amino acid incorporation programmed by MS2 phage RNA. (l), x ribosomes prepared from the polysomes by treatment with high (160 mM) NH,‘, low (3.5 mM) MgZ+, and 0.1 TIIM puromycin, in the absence of RRF and EFG; (2), m released ribosomes obtained from the polysomes in the presence of 1.18 Kg/ml RRF, 5.4 pg/ml EFG; (3), O---O high salt washed ribosomes. The values were corrected for radioactivity incorporated in the absence of MS2 RNA.

RRF addition by treating them with low (3.5 mM) Mg2+. In addition, typical ribosomes which had been washed with 1 M NH&l were prepared (washing with 1 M NHbCl is the conventional method to obtain “clean” ribosomes free of factors). Figure 21 shows that the incorporation was almost linearly dependent on the amount of ribosomes added. The activity of ribosomes released by EFG and RRF had activity comparable to those prepared by the ionic treatment. Ribosomes washed with high NH&l were much less active, suggesting that the high salt washing may damage ribosomes. We concluded that ribosomes released from mRNA by RRF and EFG are biologically active. 13. Do RRF and EFG Release Ribosomes from mRNA as 70s Single Ribosomes or as Their Subunits? Role of RRF in the ‘Ribosome Cycle” This section deals with a “reaction product” of the RRF and EFG catalyzed process, i.e., the breakdown of the termination complex. We deal with the question of whether ribosomes are released as single 70s ribosomes or as subunits. This historical question is addressed in much

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broader terms when we discuss the fate of ribosomes in the “termination complex” in Section II Subsection 2. We decided to discuss this historical problem in this section because the data regarding this information contained an important biological observation about the nature of freshly released ribosomes. This was a hot subject 20 years ago and several laboratories were engaged in active discussions regarding it (50-59). However, most of the authors involved were not aware of the fact that they were studying the reaction products of RRF and EFG at the time. Instead of reviewing many of the papers, we chose only a few papers from two seemingly opposing laboratories. The important fact which emerged from the dispute is that ribosomes produced from the termination complex by RRF and EFG are much mow prone to dissociate than 70s ribosomes isolated in a conventional manner. To indicate this fact, ribosomal subunits of freshly released 70s ribosomes are placed obliquely in the schematic model in Fig. 18. Since these results were obtained with a crude extract, the sensitivity of ribosomes being released by pure RRF and pure EFG toward concentrations of Mg’+ and the initiation factor 3 (IF3) should be examined to confirm this concept. We predict that ribosomes freshly released by pure RRF and EFG will be easier to dissociate into their subunits than the conventional 70s ribosomes. In 1970 Kaempfer (56) published a paper concluding that ribosomes are released as subunits from a messenger RNA. This is pathway B as described in Section II Subsection 2. He based his conclusion on two observations. First, under in vitro conditions in which the ribosome concentration was extremely dilute, he observed 30s and SOS subunits but not 70s ribosomes as runoff ribosomes from polysomes. An important control in this experiment was that 70s ribosomes isolated separately by the conventional method did not yield subunits under identical [Mg2+] and other conditions. Second, upon mixing heavy isotope labeled polysomes and light polysomes, he observed intermediate density 70s ribosomes under not too diluted conditions during the release of ribosomes from these polysomes. He suggested that subunits must be first formed and then reassociated during the run-off, resulting in the hybrid 70s ribosome. This was taken as strong evidence for subunits being released because exogenously added 70s ribosomes were believed to exchange their subunits only with dissociated 50s and 30s subunits. (This “belief” was refuted later as described below.) In a subsequent paper, Kaempfer indicated that released riboso-

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ma1 subunits are prevented from reassociation by IF3 (58). In this way, the separated subunits were available for the initiation process. It was found that, under the conditions where run-off ribosomes were observed as 70s ribosomes (under not too diluted polysome concentrations), addition of IF3 inhibited formation of 70s runoff ribosomes and ribosomal subunits were observed. The exogenously added 70s ribosomes which were prepared separately did not dissociate into subunits under the same conditions. This was another indication that ribosomes freshly released by RRF are different from conventional 70s ribosomes and are much more sensitive to dissociation by I F3. The view that ribosomes are released as 70s ribosomes was represented by Subramanian and Davis (25). This is pathway A as described in Section II Subsection 2. To refute the heavy-light subunit exchange experiment of Kaempfer, they mixed heavy and light polysomes and ribosome run-off was allowed under slightly higher [Mg*+] than Kaempfer. The rationale was that if heavy ribosomes and light ribosomes were released as subunits from polysomes, they must form 70s ribosomes with intermediate density. No hybrid 70s ribosomes were observed under these conditions, suggesting that RRF could release ribosomes as 70s ribosomes under their experimental conditions. This is in sharp contrast to Kaempfer’s result. They pointed out that at the low Mg*+ concentration Kaempfer used, simple mixing of heavy 70s ribosomes with light 70s ribosomes resulted in exchange of subunits. Although 9.8 mM Mg*+ used in this experiment may be on the high side, the authors mentioned that this was the optimum Mg*+ concentration for in vitro protein synthesis. At this Mg2+ concentration, RRF functions well (see Fig. 17). They also noted the fact that ribosomes being released from mRNA are more sensitive to Mg*+ and dissociate into subunits than 70s run-off ribosomes isolated from the sucrose density gradient. They proposed that at the time ribosomes are released from RNA “a conformational change takes place that would permit the subunits to open up as on a hinge while retaining jirm attachment to each other.” These experiments appear well-controlled and the conclusions seem sound. However, what actually goes on in the cell depends largely on intracellular MgZ+ concentration (60) and other conditions as described above. None of these authors’ data indicated that 30s subunits stayed on mRNA. This means that under their conditions, the termination complex did not take pathway C (Section II Subsection 2). For example, in the time course of subunit run-off under the dilute polysome condi-

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tions, Kaempfer’s data (56) showed no delay in the 30s subunits release from mRNA compared to the 50s subunits release at 1 min, 5 min, or 15 min after the onset of the reaction. If 30s ribosomal subunits stayed on mRNA under these in vitro conditions, one would expect that appearance of 30s subunits would be somewhat delayed or not observed during the appearance of SOS subunits. After describing these discussions, we should state our current view on this subject. As readers noted from Fig. 1, we observed 70s single ribosomes being released under our experimental conditions. This does not mean that we insist on the release of 70s single ribosomes as the sole outcome of the action of RRF and EFG. We believe, as described in Section II, that the outcome of the RRF action could vary depending on the environment surrounding the termination complex. The “environment” includes [Mg*+] and polysome concentrations, presence or absence of IF3, the nucleotide sequence surrounding the termination codon, and the nature of ribosomes. The important point is that RRF and EFG do “something” on the termination complex so that the “serious consequence” described in Section I Subsection 9 does not happen. This “something” includes six possible “pathways” which ribosomes may take as a result of the action of RRF and EFG as described in Section II. Therefore, we believe that both conclusions described above are correct. Even in viao, ribosomes are released as 70s single ribosomes or as their subunits depending on the environment of the polysomes. Everyone agrees that 70s ribosomes have to dissociate into their subunits sometime before the next round of protein synthesis starts. Whether this happens immediately after release from mRNA or some time later is not crucial. In connection with the above discussion of the structure of ribosomes being released from mRNA, we would like to point out the possible role of RRF in the regulation of protein synthesis. During early work on polysomes it was noted that a decrease of protein synthesis rate resulted in the accumulation of single ribosomes at the expense of polysomes while the size of the ribosomal subunit pool remained essentially unchanged (52, 61, 62). This led to the “ribosome cycle” concept in which ribosomal subunits are formed after the metabolism of the termination complex. They are, in turn, used to initiate the next round of protein synthesis. It therefore appears possible that the rate of protein synthesis may be influenced by the rate at which the termination complex is broken down. This concept probably deserves serious consideration in view of the long stay of ribosomes at the termination codon (63).

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14. The RRF Gene (frr): Location in the E. coli Chromosome, Cloning, and Expression With the advent of modem gene technology, our research on RRF naturally shifted toward molecular genetics from biochemistry and enzymology. Our first entry into this area was localization of the RRF gene position in the E. coli chromosome before the molecular cloning of this gene (64). Although gene localization, after cloning and sequencing of the gene, is a simple task, we localized the RRF gene Cfrr) before its cloning to make the cloning easy. For this purpose, we used a convenient method useful if the protein product of any gene is known on 2D gel electrophoresis. Since the method may be of general interest, we describe it briefly in this subsection. We reasoned that the diploid RRF gene would produce twice as much RRF as the haploid RRF gene. If so, by measuring the amount of RRF in various merodiploid cells we can determine which merodiploid contains the RRF gene. For quantification of RRF, total cell protein labeled with [35S] methionine was prepared from each pair of an F’-carrying strain and its F parent. Total cell protein was then subjected to 2D gel electrophoresis (65). After these measurements, we obtained the value called the normalized ratio. This value was defined as the ratio of the amount of RRF between F’ (merodiploid) and F

, O

50

0

00 Map

Position

20

40

(Minutes)

Fig. 22. Quantity of RRF in various merodiploid strains relative to the corresponding haploid strains (64). Horizontal arrows indicate the chromosomal segments carried by the F’ factors. The distance of each horizontal arrow from the xaxis represents the averaged value of the normalized ratio for two to four experiments. The standard deviations of the data are indicated by vertical bars. The open squares on some of the arrows indicate that the data were obtained from a single experiment.

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(normal control). As shown in Fig. 22, merodiploid #42.51 had a higher RRF content than others, while merodiploid #42.50 did not have a high RRF content. Since F’ of 4251 covers 97 to 6 min and that of #4250 covers 97 to 2 min, we concluded that the gene for RRF must be located at 2 to 6 min on the E. coli chromosome. After localizing the RRF gene on the E. coli chromosome, we found it in pLC 6-32 (covers 4 min region) of the Clarke-Carbon Gene Bank with the synthetic DNA probe prepared according to the partial amino acid sequence of RRF. During these studies, it came to our attention that Bendiak and Friesen had reported earlier cloning of the 4 min region of the E. coli genome (66). To our surprise, their 2D gel showed a 22 Kd protein that behaved like RRF! They did not realize that the protein might be RRF and did not pursue this protein further. The exact localization of the RRF gene on the E. coli chromosome was then relatively easy because the 4 min region contains the gene for peptide elongation factor Ts (EFTs, tsf gene). While sequencing the region surrounding the RRF gene, we identified the EFTs gene nearby. Thus, we determined the exact location of the initiation site for RRF to be 1.1 kilobases downstream from the translational termination site of tsf. Now that the gene was localized, we wanted to call this gene rrf but this was already taken for another gene and therefore the gene was called frr (factor for the release (recycling as the name changed) of ribosomes from mRNA). The nucleotide sequence and deduced amino acid sequence showed (31) that RRF consists of 185 amino acids and its calculated molecular weight was 20,639, which was in agreement with our experimentally determined molecular weight (18,000 by the gel filtration method and 23,500 by SDS-PAGE). Sequencing the region downstream of the RRF gene revealed the rho-independent transcription termination signal. RRF has the usual SD sequence and the initiation codon GUG. The amino acid sequence of RRF will be discussed in Section I Subsection 17. A pleasant surprise for us was that pRR1 which is a pUC19 plasmid containing the 2.2 kb fragment harboring frr expressed this protein so well that RRF was the major protein in the host cell of this plasmid. Figure 23 shows the PAGE analysis of the total protein extracts of E. coli harboring pRR1. As can be seen, RRF was one of the major proteins in this extract. This over-expression of RRF only slightly inhibited the host’s growth rate. The fact that RRF can be obtained in quantity from this strain is an important point for our

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A

Loa 66.0

1

2

3

4

5

-

$1; 24:0 20.1 14.2

-

I I 29.0 24.0

I 20.1 KDa

Fig. 23. Expression of RRF by E. coli DHW harboring pRR1 (69). Plasmidcarrying cells were disrupted by freeze-thawing and cell debris was removed by centrifugation. The resulting ceil extracts were subjected to 15% SDS-PAGE. Lanes: 1 and 5, molecular weight marker proteins; 2, cell extracts of DHSa harboring pUC19 (6 gg); 3, cell extracts of DHSa harboring pRR1 (6 pup); 4, partially purified RRF (920 ng). The arrow indicates p-lactamase.

Fig. 24. Least-energy folding model of RNA containing the frr coding region (31). The DNA sequence shown in Fig. 26 was used for processing by the program of Zuker and Stiegler (67), and the results were redrawn. Possible nucleotide base pairs are connected by a bar; a dotted line represents nucleotides which are connected but cannot be drawn adjacent to one another due to the presence of a loop.

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future structural studies by X-ray crystallography and NMR which require a relatively large amount of easily soluble protein. The exact reason for the efficient expression is not known, but two reasons are conceivable. First, as described in the next section, the promoter for RRF is very strong (comparable to the Zac promoter). Second, the mRNA may assume a stable structure and therefore may have a long half-life. Figure 24 shows a model of the RRF mRNA structure drawn with the Zuker-Stiegler program (67). Approximately 60% of the 7.55 nucleotide sequence can be paired in this model. Immunoglobulin heavy chain mRNA and tRNA have 56 and 54% pairing, respectively. If the possible pairing has anything to do with the stability of mRNA, mRNA for RRF may be as stable as these RNA. It would therefore be of interest to measure the half-life of RRF mRNA. 15. Characterization of the RRF Promoter An interesting feature of the frr promoter was revealed when the upstream region of frr was examined. Since RRF is an essential protein (see next subsection), which participates in protein synthesis, there is a strong possibility that the expression of frr may be regulated through transcriptional control depending upon environmental conditions. As a first step toward understanding the control of frr expression, we characterized the promoter region of this gene. To investigate the properties and precise location of the promoter region, we subcloned the RRF promoter region into the promoter-cloning vector, pKK2328. In this system described by Brosius (68), DNA fragments containing promoter activity are inserted upstream of the promoterless gene for chloramphenicol acetyltransferase which confers chloramphenicol resistance (Camr). Thus, promoter activities were measured by both CAT activity and the expression of the Cam’ phenotype. Comparison of the promoter activity of frr with that of the Zac promoter revealed that the former is slightly more acitve than the latter (69). Various deletions were introduced into the promoter region of RRF and the loss of the promoter activity was monitored. The results shown in Table IX defined the promoter region as described in Table X. Comparison of the -35 region of the RRF promoter and that of the E. coli promoters containing the DNA sequence TATAcT in the -10 region is shown in Table X. The RRF promoter is unusual in that it has unusually long 20 bp spacer. Out of 263 promoters in E. cob (70) only one promoter with a 20 bp spacer was identified through a literature survey (71). The rest were shorter.

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TABLE IX CAT Activities

and Camr

Levels

from

Nested

Deletions

cloned

into

the Vector

ET AL.

pKK232-8

(69) DNA

Plasmid pKKR1 pKKR2 pKK52B pKK35B pKK36B pKK55B pKK49B pKK41 B pKK60B pKK58B pKKR1 U The pKK plasmids were with varying numbers of pKK232-8 promoter-fusion deleted together with 139 CAT activity. Cam’ levels plates supplemented with

fragment inserted

CAT specific activity (U/mg of protein)

Cam’ level Wml)

+12/-co.1440 + 12/- 138 + 12/- 116 + 12/- 100 + 12/- 85 +12/-80 +12/-71 +12/-54 +12/-44 +12/-29 - ca.1440/ - 139

60.3 58.9 58.8 61.0 30.6 28.4 26.5 0.4 0.5 0.4 0.4

500 500 500 500 30 30 30 2 2 2 2

obtained by cloning the first 12 nucleotides of the frr cistron (32) upstream nucleotides (expressed as negative numbers) into the vector. In plasmid pKKR1 U, the frr-specific 12 nucleotides were upstream nucleotides. Cell extracts were prepared and assayed for were determined by streaking liquid cultures on Trypticase soy agar various amounts of chloramphenicol.

TABLE X Comparison of the RRF Promoter TATAcT in the - 10 Region (69) Promoter

-35

Consensus RRF

and E. coli Promoters

region

ITGACA

Spacer 17 20

Containing

-10

the DNA

Sequence

region

TATAAT

.acgaTTacCcgtaatatgtttaatcagggcTATAcTtagcac~ctt..

lt%XA rpmB-rpmG Ml

RNA

&A grid

rpsu Pl rpoD Pa deo P3

I 100 TTccaA TTGAgc gTGACA cTGttA TTtAtA TcGcCc caGctA TcGcCg

I - 80 17 17 17 17 17 17 17 16

I - 60 TATAcT TATAcT TATAcT TATAcT TATAcT TATAcT TATAcT TATAcT

The DNA sequence of the RRF promoter region is shown between positions -100 and -55. The transcriptional initiation site is indicated by double underlined a at position - 58. Nucleotides in capital letters indicate those homologous to the consensus sequence (I 75-l 77). E. colt’ promoter sequences containing TATAcT in the -10 region were obtained from the following sources: ZexA (178); rpmB-rpmG (179); Ml RNA (180) gZyA and grid (175); rpslJ Pl (f81); rpoD Pa (182); deo P3 (183).

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Whether or not the unusual spacer length plays a role in the control RRF expression remains to be seen.

163 of

16. Essentiality of RRF for Bacteria and In Vivo Function of RRF 1) Gene knock-out and use of temperature sensitive plasmid for carrying wild- type RRF Since the discovery of RRF came from in vitro biochemical studies, it is of utmost importance to demonstrate what we observed in vitro in living bacteria. This is especially important for RRF because its action, the release of mRNA from ribosomes, can be observed without RRF in vitro if Mg2+ concentration is lowered below 3.5 mM. In our most recent publication, we demonstrated the essentiality of RRF for bacterial growth (28). In this subsection, we describe this experiment briefly. We inserted a cytidine base into the frr coding frame at position 259 (counting from the first base of the initiation codon of the RRF coding sequence). This frameshifted RRF gene was placed in a plasmid whose replication is temperature sensitive (pMAK705 (72)). This plasmid gives E. coli resistance to chloramphenicol. When the bacteria harboring this plasmid are grown at 42°C in the presence of chloramphenicol, the plasmid is integrated into the host chromosome because the plasmid cannot replicate at this temperature. Upon shifting to the permissive temperature, however, the plasmid often comes out of the chromosome. In this process, the wild-type RRF gene may often be excised with the plasmid while the mutated gene stays in the chromosome. Sequencing of the RRF gene both in the chromosome and in the plasmid did verify this gene exchange. The host bacteria with the mutated RRF must depend on the presence of pMAK705 which contains the wild-type RRF if the RRF gene is indispensable. As the plasmid is temperature sensitive, the bacteria should then be temperature sensitive. If the RRF gene is dispensable, the bacteria will not be temperature sensitive and one can therefore determine the essentiality of RRF. The results of such an experiment are summarized in Fig. 25. As shown in panel B of this figure, viable counts of MC1061-2 (mutated fw in the chromosome, wild-type fw in the temperature sensitive plasmid) stopped increasing after a 2 hr lag following the temperature shift-up, even in the absence of chloramphenicol (CM, open square symbol). In contrast, MC1061-1 (wild-type frr in the chromosome, mutated frr in the temperature sensitive plasmid) showed no such temperature-sensitive growth (panel A). Furthermore, MC10611 which grew at 43°C in the liquid culture could not form colonies in

164

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ET AL.

lo9

lo9

lo8

lo*

10'

10'

ij

106

106 0

2

4

6

8 Time

10

0

2

4

6

8

lo

(hours)

Fig. 25. Temperature sensitive growth of E. coli containing frame-shifted, nonfunctional frr in the chromosome and wild-type frr on temperature sensitive CM’ plasmid (28). A: E. coli MC1061-1 carrying wild-type frr in the chromosome and mutated frr in its temperature-sensitive plasmid. B: E. colt’ MC1061-2 carryplasmid and mutated frr in the bacteing wildtype frr in its temperature-sensitive rial chromosome. Bacteria were grown in Luria broth (LB) without any antibiotics and plated onto LA plates with or without chloramphenicol (CM) at 32°C. Symbols: V, growth at 32°C and plating onto non-selective agar; v, growth at 32°C and plating onto CM plates; 0, temperature shift from 32°C to 43°C at 1 hr in LB and plating onto non-selective agar; n , temperature shift as above and plating onto CM plates.

the presence of CM (closed square symbol). This indicates that the plasmid loss at 43°C in the liquid culture took place equally with MC1061-1 and MC1061-2. Therefore, the cessation of growth of MC1061-2 at 42°C in the absence of CM was due to the presence of mutated frr in its chromosome. In the control experiments at 32”C, both strains behaved identically (triangle symbol). We concluded that frr is essential for bacterial growth. 2) Use of incompatible plasmids for proving the essentiality of RRF We have obtained additional evidence for the essentiality of the RRF gene. The principle of this experiment depends on the use of incompatible plasmids. Certain plasmids cannot coexist within the same host bacteria for long, and these are called incompatible plasmids. On the other hand, under certain conditions where the host bacteria must contain both plasmids, bacteria retain these incompatible plasmids to survive. Thus, if all surviving bacteria harbor both incompatible plasmids, these plasmids must contain essential genes. This system therefore tests for the essentiality of genes carried by incompatible plasmids.

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TABLE XI Essentiality of frr

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Overcomes

Plasmid

Incompatibility

(28)

Colonies Line

1 2 3 4 5 6 7 8 9 10

Hosta

Resident plasmidb

MC1061

pMAK705

MC1061-2

pMR2

MC1061-1

pMR2M

MC1061

pMR2

MC1061-2 MC1061-2

pMR; pRR2 pMR2; pRR2M

Incoming plasmid

pMWll9 psc101 pMWl19 psctot pMWl19 pSClO1 pMWl19 pSClO1 psc101 pSClO1

with

Selection for resident plasmid 0 0 0 0 0 0 0 0 0 0

both incoming plasmidsC (%) Selection for incoming plasmid 0 0 100 loo 0 0 0 0 0 100

and resident

NO

selection 0 0 0 0 0 0 0 0 0 0

(iSO/rSO) (i94/r6) (r100) (r100) (i81/r19) (i94/r6) (i56/r44) (i94/r6) (i56/r44) (rloo)

’ All strains are MC1061 derivatives. MC1061 and MC1061-1 carry wild-type frr in their chromosomes, but MC1061-1 also contains the pMR2M plasmid with mutated frr. MC1061-2 carries mutated frr in its chromosome and wild-type fir in its pMR2 plasmid. “pMAK705, pMW119, and pSClO1 all carry the pSClO1 replicon and code for CM, ampicillin, and tetracycline resistance, respectively. pMR2M is pMAK705 containing an 893-bp Smal-EcoRI insert carrying the promoter, the frame-shifted coding sequence, and the rho-independent transcription terminator forfrr. pMR2 is identical to pMR231, except that it carries the wild-type frr. pRR2 and pRR2M are pUC19 derivatives coding for ampicillin resistance and carrying the same insert as pMR2 and pMR2M, respectively. ‘ Bold-faced characters indicate alteration from the expected incompatibility results. Numbers in parentheses indicate the percentage of colonies carrying the incoming (i), or the resident (r) plasmids when single-colony isolations were made in the absence of selection.

The result of such an experiment based on the principle stated above clearly showed that RRF is indispensable. In the experiment described in Lines 1 and 2 of Table XI, the CM-resistant plasmid pMAK705 (the vector forfrr in this study) was shown to be incompatible with both the ampicillin (AP)-resistant plasmid pMW119, and the tetracycline (TC)-resistant plasmid pSClO1. The experiments described in Lines 3 and 4 show that, despite this incompatibility, all of the strains retained the resident CM-resistant plasmid pMR2 (containing the wild-type frr) when the host chromosome had the mutated frr (MC1061-2). Bacteria could not survive without plasmid pMAK705 which contains wild-type frr. The control MC1061 -1, carrying the wild-type frr in its chromosome, expressed regular incompatibility as is shown in Lines 5 and 6. The incompatibility was masked in pMC1061-2 under the selective condition for the incoming plasmid. This was not due to the possible

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abnormal nature of pMR2 (resident plasmid), because this plasmid was readily segregated from the incoming incompatible plasmids in the MC1061 strain that carried the wild-type frr in its chromosome (Lines 7 and 8). In addition, the masking of the incompatibility due to the presence of RRF was not due to a change in MC1061-2 host functions, because, as shown in Line 9, expected incompatibility was observed if the strain carried an additional compatible plasmid, pRR2, with the wild-typefrr insert. Under the same conditions, if the compatible plasmid carried the mutatedfrr (pRR2M), pMR2 could not be eliminated (Line 10). We concluded that frr is essential. 3) Temperature sensitive mutant of RRFin vivo function of RRF An in vivo functional test of fry has recently been carried out with the use of temperature sensitive mutants. We have isolated 18 temperature sensitive mutants of frr (to be published elsewhere). The long-awaited experiment to obtain evidence that loss of RRF activity leads to reinitiation of translation in vivo has finally been carried out with our tsfrrl4. In this experiment, we placed a plasmid containing the DNA coding for the fusion protein of the Zac I gene and the P-galactosidase (73), kindly supplied by Dr. D. A. Steege, Duke University. The particular fusion construct we used had the ochre mutation on the 6th codon of the I-gene. This UAA codon is in-frame with the P-galactosidase reading frame. When E. coli tsfrrl4 harboring this plasmid was grown at 32”C, there was no P-galactosidase activity detected because of the UAA codon at the 6th codon. However, when the culture temperature was switched to 42°C a time dependent steady increase of /3-galactosidase activity was observed although cell growth stopped due to the inactivation of temperature sensitive RRF (to be published elsewhere). Although we have not yet characterized the newly synthesized fusion protein, we expect that this protein has the amino terminal amino acid corresponding to the 7th codon of the I gene. An important control, where UAA is out of the reading frame of /3-galactosidase, has yet to be performed. At any rate, this experiment represents the first demonstration of translational reinitiation in vivo upon specific inactivation of RRF.

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17. Amino Acid and DNA Sequence of E. coli RRF in Comparison with Recently Published Sequences of Similar Proteins At the time the complete sequence offrr (shown in Fig. 26) was determined in our laboratory, no similar sequences were known. However, c AGG GCT ATACTTAGC *c* CTTCCA CT6TGTGTG ACTGTC TGG TCTGAC C-76) ftl ttc

TCA GAC AAG (-21) (8) (t) ctg cgn ctt GAT GCT GM ASP ALA GLU ----------__---^_-_--

TTT

TCA AGG ATT

COT MC

GTG A,T AGC OAT ATC YET ILE SER ASP ILE _---_________------------

&GA AAA ARG LVS

(211 7

“’ ca GTA “AL

CCC ATG GAC AM ARG MET ASP LYS

KC CM THR GLN

(69: 23

ATA ILE

CCC ACG GGT CGT GCT TCT CCC AGC CTG CTG GA1 GGC ARG THR GLY ARC ALA SER PRO SER LEU LEU ASP GLI

I1171 39

ATC ILE

AGC AA* SER LVS

ATT ILE

GTC GTG GAA TAT TAC “AL V&L GL” TVR TVA

AGC GTA KG GTA GAL GAT SER “AL THR “AL GLU ASP

TGC GTA GM CYS “AL CL”

GCG TTC AM ALA PHE LVS

GGC AC0 CCG ACG CCC CTG CGT CAG CTG GCA GLY THR PRO THR PRO LEU ARG GLN LEU ALA

(165) 55

TCC CGT ACA CT0 AAA SER ARG THR LEU LYS

(213) 71

ATC ILE

AK GTG TTT GAT ASN “AL PHE ASP gac CT1 LEU

(2611 67

GGC CTG AAC CCG AK TCT GCG GGT AGC GAC ATC CC1 GTT CL” LEU ASN PRO ASN SER ALA GLV SER ASP ILE ARC YAL __________-----_-__----------------------------------------

CCG CTG CCC PRO LEU PRO

(309) 103

CCC CTG ACG GAA GAA CGT CGT AAA PRO LEU THR GLU GLU ARC ARG LYS -v--------v

GTT “AL

CGT GGT ARG GLV

(3511 119

GCA GTA CGT AAC GTG CGT CGT GAC GCG ALA “AL ARG ASN VAL ARG ARG ASP ALA

I4051 135

:GT TCA ATG TCT CCC CCC GTT GAA AAA CCC ATT ARG SER MET SER PRO &LA “AL GLU LVS ALA ILE

GAA GCA GM CM GCG CGT GTT GLU ALA GLU GLN ALA ARC “AL MC AS,,

UC cgc ags ctc ATG GCG TCC CAT MET ALA SER ASP

GAT CTG ACC &AA ATC ASP LEU THR LVS ILE

GAC .,A., GTG AAA GCA CTG TTG AAA ASP LYS “AL LYS ALA LEU LEU LYS

GAT AAA GAG ATC kGC GM GAC ASP LYS GLU ILE SER GLU ASP

(453) 151

CTG ACT CAT GCT GCA LEU THR ASP ALA ALA

(501) 161

GAA GCG GCG CTG GCA GAC AAA GM GCA GAA CTG ATG GLU ALA ALA LEU ALA ASP LVS GLU ALA GLU LEU MET

(5491 183

CAG TTC TGA TTT GLN PHE STOP

CTT

I591 1 186

QAT COG w

GCC TTT

TGC TTT

CTC KC

ATT

GAC CAT ASP ASP ATC ILE

TTC

CCC CGT TCT CAG GAC GA1 GTA CAG AAA ARG ARG SER GLN ASP ASP “AL GLN LVS

AAG AU LYS LVS

AT0

MC

ATT ILE

CM

GAA CGA CU

TCT

GGA TGT

(645,

CTG GGC TCG ACC GGC TCG ATT

GGT TGC

(693)

CGC GTA GTT

(741!

TTA

AGC ACG CTG GAC GTG GTG CGC CAT MT GCG CTG GTG GCA GG

TTC

TGT CTC AK

CCC GM

CAC TTC

(7551

Fig. 26. Nucleotide sequence and deduced amino acid sequence of the fw gene (31). The nucleotides are numbered, in parentheses, from the initiation site (GTG) of the 185 amino acid open reading frame. Numbers without parentheses indicate amino acid numbers starting with the initiator methionine. The underlined region indicates a rho-independent terminator of transcription. The broken lines indicate the amino acid sequence determined from the NHz-terminal analysis of cyanogen bromide-cleaved peptides of RRF. Nucleotide sequences shown in lower case letters indicate the probes synthesized according to the sequence of the cyanogen bromide-generated peptide. Sau3AZ and BglZZ sites which were used for sequencing are also indicated. “....” indicates the presumed Shine-Dalgarno sequence.

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L. JANOSI ET AL. 1 ECORFtF HINRRF MGERRF NLP

50 FiTQI LKGHI KAHYIDFF KQAADKKIQW LKEEL KQ...... . . . . . . . . . . VSTPL

ECOF7RF HINRRF MGERFS

NLP

ECOFtRF HINRRF NLP

ECORRF HINRRF LENSLKA EI NLP Fig. 27. Comparison of amino acid sequences of bacterial RRFs and a glycine rich nuclear protein from carrot. Deduced amino acid sequences of RRF from E. coli (ECORRF (31)), the putative RRF of H. injfuenzae (HINRRF (75)) and M. genitalium(MGERRF (I)) and the glycine rich nuclear protein D2 from Daucus carota (NLP, GenBank accession number X72384 (76)) are aligned to emphasize homology. Alignment was made with the hierarchical clustering method (188) using the amino acid substitution matrix of Risler et al. (74). Identical or interchangeable amino acids in all four sequences are boxed. Positions of identical or interchangeable amino acids present in three sequences are indicated by an “0” symbol above the sequence. Shaded areas indicate locations of sub-sequences showing similarity (but not identity) to known motifs or sequence signatures as identified by the Motifs program of the GCG software package (Genetics Computer Group, Inc. (78)). The motifs identified are: in ECORRF, G-protein coupled receptors signature (from T49 toT64); in HINRRF, ATP/GTP-binding site motif A (from A9 to S16 and from G119 to V126) and GTP binding elongation factors signature (from D86 to 198); in NLP, ATP/GTP binding site motif (G28 to S3S). Note that amino acid positions and alignment positions are not necessarily the same, because of the introduced gaps, which are indicated by a “. ” symbol.

during the past six years since the cloning offer, many prokaryotic and eukaryotic genes have been sequenced. In this section, we discuss the sequences of RRF-like genes in other organisms. In MycopZusma genitalium (I) a highly similar gene was found and

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TABLE XII Comparison of DNA

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Sequences

of RRF

Related

Proteins 2nd sequence

1 st sequence

_Name Ecorrf Ecorrf Ecorrf Hinrrf Hinrrf Mgerrf

From...To 2-555 SOS-548 233-515 468-545 233-549 385-549

(bp)

Name Hinrrf Mgerrf D2 hlgerrf D2 D2

F rom...To 2-555 499-542 179-461 462-539 179-495 325-501

(bp)

Homology (%) 67.7 63.6 57.5 65.4 54.9 60.0

Nucleotide sequences of RRF-coding genes from E. coli (Ecorrf, (32)), H. in~%enzae (Hinrrf, (7.5)) and M. genitalium (Mgerrf, (f)), and the gene for the glycine-rich nuclear protein D2 from carrot (D2, GenBank accession number X72384 (76)) were compared by the BestFit program of the GCG package (Genetics Computer Group, Inc. (78)).

identified as RRF. The deduced amino acid sequence of this gene was 34% identical to E. coli RRF and 57% similar according to the algorithm described by Risler (74) (Fig. 27). The homology at the nucleic acid level was 64% (Table XII). The fact that M. genitalium’s “RRF” is nearly identical in length (184 amino acids) to E. coZz’)sRRF (185 amino acids) is very significant, since the genome size of E. coli is almost ten times larger than that of M. genitalium. Whereas this organism manages without RF2 and RF3 (I), it retains RRF, suggesting the importance of RRF for bacterial life. Since M. genitalium has the smallest known genome of any free-living organism, RRF must be one of the minimum requirements for life. In accordance with this view, an RRF-like gene was also found in the Huemophilus in&enzue genome (75) (Fig. 27). E. coli RRF and the deduced amino acid sequence of the H. injkenzue gene were found to be 69% identical and 86% similar (74). Th e indispensability and importance of RRF for prokaryotic organisms are again suggested by the fact that H. injluenxue “RRF” has a length identical to E. coli RRF although H. influenxue has a genome length only one third of that of E. coli (75). During our search for sequences similar to that of E. coli RRF, we came upon a surprising finding. As shown in Fig. 27, a cDNA sequence identified in carrot tissue culture called D2 (called DCD2 NLP in the GenBank) has a 45% direct homology and a 65% similarity to E. coli RRF. Schrader et al. (76) were interested in the gene expression during embryogenesis of carrot cells. When the plant hormone auxin was re-

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moved from the in vitro carrot cell culture, the mRNA having a sequence similar to RRF steadily increased up to 20 days after the removal of auxin. The increase of this mRNA was as much as 100 fold and this high level was maintained during the course of this experiment. Although the function of this protein is unknown, Schrader et al. suggest that it may be a nuclear protein (NLP) because a “bipartite nuclear location” sequence is present between the amino acid residues 114 and 130. This sequence is a combination of two short stretches of basic amino acids separated by a group of more than four residues. The basic amino acids are RR (114th and 115th amino acids) and KK (128th and 129th amino acids) as identified in NLP. The corresponding sequence in both E. coli and H. inJEuenzae RRF would be RR (133rd and 134th amino acids) and K (145th amino acid). This motif presumably forms a signal sequence conferring the import of proteins into the cell nucleus (77). Whether or not these sequences have biological significance remains to be seen. The stretch of amino acids T49 to T64, which are highlighted, is the motif common to G-protein coupled receptors (78). This is significant in view of our conclusion that RRF and EFG must work simultaneously for release of ribosomes from mRNA (Section I Subsection 11). In addition, amino acids D86 to 198 of HINRRF represent the motif common to proteins involved in peptide chain elongation (78). This further strengthens our belief that RRF is indeed a factor in protein synthesis (Section I Subsection 10). RRF may also be involved in error prevention during translation as discussed in the following subsection. Since error prevention is performed every time the peptide chain is elongated, this motif fits well with our preliminary data. Table XII shows the comparison of the DNA sequence of RRF and RRF-like proteins. Between 60-70% homology was observed with these DNAs coding for the proteins mentioned above. 18. Other Functions of RRF 1) Functions directly related to the release of ribosomes from the termination codon Thus far, we have discussed RRF solely as a factor that releases ribosomes from mRNA at the termination codon. In this subsection, we discuss our preliminary data and data from other laboratories which indicate that RRF may have other functions. First, in addition to the ribosomes at the termination codon, our data indicate that any ribosomes which have accidentally lost peptidyl groups during the chain elongation step would be released by RRF and

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EFG. This is obvious from the fact that RRF was discovered through the use of the model substrate, puromycin-treated polysomes. Second, although we do not have direct evidence, we can presume with reasonable certainty that RRF and EFG can remove elongating ribosomes which have lost peptidyl tRNA. This is because the absence of tRNA on the ribosome will make it even easier to release ribosomes from mRNA. This possibility that ribosomes may lose growing peptidyl tRN‘4 is perhaps not too remote because E. coli is equipped with an enzyme which hydrolyzes peptidyl tRNA, peptidyl tRNA hydrolase (79). It has been shown that lack of this enzyme is lethal (80) for E. coli suggesting that release of peptidyl tRNA may happen frequently and that free peptidyi tRNA is toxic. Removal of ribosomes without peptidyl tRNA from mRNA may be important to prevent wasteful synthesis of non functional proteins with portions of NH>-terminal ends missing. 2) Error prevention In addition to the release of ribosomes from mRNA, we have a preliminary indication that RRF may function to prevent translational errors. It is known that the E. coli in vitro protein synthesis system makes occasional errors. In polyphenylalanine synthesis programmed by polyuridylic acid, the occasional incorporation of Ile into The misincorporation of Ile into polyphenylalanine takes place. polyphenylalanine in this system is stimulated by streptomycin (81) through its effect at the step of codon specific binding of aminoacyl tRNA to ribosomes (5). We found that the background misincorporation of Ile into polyphenylalanine in the absence of streptomycin was significantly stimulated if we remove RRF from the in vitro protein synthesis system (to be published elsewhere). This observation made by Dr. R. D. Ricker in his Ph. D. dissertation (82) should be extended and the mechanism through which RRF may exert such a role for preventing misreading should be elucidated. One possible mechanism for the error prevention function of RRF is that RRF may bind to the A site of the ribosome because it may have an affinity to this site like other elongation factors such as EFTu (20). T o p er f orm the role of error prevention, RRF has to have a greater affinity for the A site than noncognate aminoacyl tRNA but less than cognate aminoacyl tRNA. Thus, RRF may occupy the A site while the ribosome looks for the cognate aminoacyl tRNA so that noncognate aminoacyl tRNA will not bind to the A site. The fact that crude ribosomes isolated from E. coli contained a lot of RRF ((26) and the following paragraph) may be consistent with this notion.

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Additional evidence for the notion that RRF may be an error prevention protein is its abundance in E. coli cells. Immunological quantification with an antibody against pure RRF according to the method of Heremans (83) revealed that about 8,000 RRF molecules exist per cell (84). This corresponds to more than one third of the number of ribosomes in an E. coli cell (85). Thus, RRF is a fairly abundant protein. If its sole function is to release ribosomes from mRNA at the termination codon for “recycling,” E. cob would not need this much RRF because one would assume that only a fraction of the total ribosomes is engaged in the termination step.

3)

RRF as a transcriptional stimulator

Is there a possibility that RRF is involved in a completely different biological activity other than translation? The suggestion that RRF may indeed have additional roles came from our preliminary experiments dealing with the subcellular localization of RRF (84). The finding that the ribosomal wash contained a significant amount of RRF (26) prompted us to determine which fraction of the cell extract contains the most RRF. From 1.6 g of wet E. coli, 13 units, 17 units and 3 units of RRF were recovered from SlOO, the intermediate layer and ribosomal wash, respectively. Since the intermediate layer contains membrane fragments and partially degraded DNA fragments, RRF could be bound to DNA in viva and might have something to do with transcription. Alternatively, RRF may be associated with the RNA polymerase complex which is also found in the intermediate layer (86). However, any basic protein such as RRF associates with the “intermediate layer”, and localization of RRF in this layer may not have any biological significance. More convincing evidence that RRF is indeed a stimulatory factor for transcription was provided by the report of Kung et al. (26). In their DNA dependent in vitro protein synthesis system, they showed that RRF stimulated [3H] UTP incorporation into RNA by crude E. coli extract about two fold. In this same system, RRF stimulated pgalactosidase synthesis about four fold. In a separate experiment, they state that RRF stimulated UTP incorporation by purified RNA polymerase two fold. These results suggest that RRF in E. coli may be localized in the so-called “nuclear body” where replication of DNA and transcription of RNA takes place. Interestingly, the eukaryotic “RRF-like” protein, carrot D2 (76) appears to be a nuclear protein. An example of a translational factor involved in RNA synthesis of Q/3 phage has already been

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RRF

documented (87). We should therefore consider other biological of RRF in addition to its crucial role in protein synthesis.

II.

METABOLISM

TIONAL

COUPLING:

OF THE

TERMINATIOK

A POSSIBLE

ROLE

COMPLEX

AND

roles

TRANSLA-

BY RRF

1. Introduction Although we documented in Section I that RRF and EFG release ribosomes at the termination codon from mRNA (37) and from the model substrate (puromycin-treated polysomes), a review of the literature shows that termination might not always be followed by the complete release of ribosomes. These observations are related to translation reinitiation and coupling. Reinitiation of translation within a cistron following a premature termination codon occurs in prokaryotes (42, 73) and probably in lower eukaryotes (88) as well. Translational coupling in which translation of a downstream cistron depends, to varying extents, on translation of its upstream cistron has been observed in bacterial plasmids (89-92), IS elements (93), bacteriophages (9697), several bacterial operons (98112) and in chloroplast (113). W e wish to discuss these observations in this section in light of the RRF function. It should be noted that in uncoupled regular translation, no one doubts that ribosomes must leave mRNA at a certain point. Even in the cases discussed in this section, with few exceptions, only a fraction of the ribosomes continue to translate the downstream coupled gene. We are, therefore, discussing rare cases where ribosomes may not leave mRNA. This is by no means a review of translation reinitiation and coupling. Therefore, only a few examples of these observations are discussed. We focus on those reports which studied or proposed some kind of mechanism for reinitiation or translational coupling. In addition, we further restrict this review to those systems in which an experimental approach for studying the involvement of RRF in these processes could be carried out relatively easily. 2. Fate of the Ribosomes in the Termination Complex Before we begin the discussion of cases of translational coupling and reinitiation, we wish to consider various possibilities for the fate of ribosomes and their subunits after the release of the peptidyl group. In the following discussion, the “termination complex” refers to the complex as shown in Fig. l-11 (middle diagram).

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A) Ribosomes are released from mRNA as 70s single ribosomes and not used for coupled reinitiation. This is the situation we discussed throughout Section I. As discussed in Section I Subsection 13, this mechanism was favored by Davis’ group and others. B) Ribosomes are released from mRNA as their subunits and not used for coupled reinitiation. This is the pathway supported by Kaempfer and others as discussed in Section I Subsection 13. C) The SOS subunit is released from the termination complex first. The 30s subunit which remains transiently on mRNA but is eventually released, then initiates at the downstream initiation codon. This mechanism is supported by the data assembled by Martin and Webster (315) as described in Section II Subsection 4. D) The 50s subunit is released from the termination complex first and the 30s subunit remains on the mRNA. The 30s subunit then starts scanning up and down on the mRNA until it finds a proper place to start another round of protein synthesis. This view is favored by van Duin’s group (I 16) as discussed in Section II Subsection 9. E) The large subunit (60s) is released from the eukaryotic termination complex first and the small subunit (40s) remains on the mRNA. The 40s subunit starts scanning only toward the 3’ direction until it meets the proper nucleotide sequence for the next round of protein synthesis. This appears to be the case in eukaryotes. Kozak and others support this scenario as discussed in Section IV Subsection 2. F) The same as A and B but the released ribosomes are used for translational coupling or reinitiation. Local high concentration of freshly released ribosomes allows them to bind to the next initiation codon. This pathway is supported by the fact that the distance between the termination site and the reinitiation site is relatively short in prokaryotes. If it is not short, RNA configuration or a short open reading frame in between the termination and the reinitiation sites makes the actual distance short. In fact, the translational coupling in prokaryotes is usually better when the distance is artificially shortened (see Section IV Subsection 2). These observations are also consistent with pathway C. G) The 70s ribosome moves from the termination site toward the 3’ end without peptide synthesis until it meets the initiation site of the next cistron. This is the classical “phaseless” movement of ribosomes. Though not completely excluded, our result discussed in Section I Subsection 9 indicates that this pathway is unlikely. Any of the pathways A through G can occur simultaneously. In other words, at a certain termination codon, the fate of ribosomes is not limited to one pathway.

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3. Our Hypothesis Our hypothesis specifically denies G, but is compatible with all others. We postulate that, regardless of the pathways discussed above (A-F), RRF and EFG are always involved in breakdown of the termination complex. Pathways A through F may happen depending on the environment of the termination complex, which includes the nature of the subunit (40s or 3OS), concentration of ribosomes, local [Mg’+], the presence or absence of IF3 and nucleotide sequence surrounding the termination codon. This hypothesis, therefore, expands on the function of RRF and EFG from the simple release of 70s ribosomes to catalysis of part or all of the pathways described above. For example, \ve postulate that RRF and EFG are responsible for the dissociation of ribosomes into their subunits at the termination codon to accommodate pathways B through F. IF3 catalyzes the dissociation of 70s subunits (114). However, IF3 has not been shown to dissociate 70s ribosomes into subunits at the termination codon, leaving 30s subunits behind on the mRNA. In fact, this task cannot be performed by IF3 because IF3 would release the 30s subunit and tRNA from mRN4. Drs. Gualerzi and Pon found this peculiar but important action of IF3 in our laboratory (6). IF3 is known to guide the 30s subunit and fmet-tRNA to the initiation site through the help of the SD sequence (for review see (114)). In contrast, IF3 breaks down the complex of the 30s subunit, tRNA and the mRNA if the complex is not the initiation complex. We believe that this action of IF3 is nature’s device to remove the unwanted 30s subunit-mRNA complexes and leave the physiological initiation complex intact. This observation was later confirmed by Martin and Webster who observed release of 30s subunits from mRNA by crude initiation factors (Table II of (215)). Having ruled out the possibility that IF3 dissociates the SOS subunit from the termination complex, we postulate that EFG and RRF perform this task under certain circumstances. There are three sets of experimental data which are consistent with this hypothesis. a) With RRF present, ribosomes do not translate the portion downstream of the termination codon, while in the absence of RRF they do. This observation is consistent with the complete release of 70s ribosomes from mRNA as well as with the preferential dissociation of the 50s subunit from the termination complex. No translation would occur with the 30s subunit alone on the mRNA. b) The 30s subunit is transiently left behind on mRNA of f2 RNA phage presumably at the termination codon of the coat cistron in the reaction mixture which

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should contain RRF and EFG (115). c) As discussed in Section I Subsection 13, there is convincing evidence that freshly released ribosomes are prone to dissociate into subunits. In fact, under certain conditions only subunits were observed (pathway B). Our model does not preclude the possibility that some exogenous ribosomes may participate in the translation of the downstream cistron. Thus, it is open to the possibility that ribosomes translating the upstream cistron may open up the occluded downstream cistron to make it available for exogenous free ribosomes. Our model neither supports nor denies the possibility that the flow of ribosomes passing through the initiation codon out-of-frame may inhibit exogenous ribosomes from initiating at that codon. 4. In Vitro Translation of RNA from RNA Phage This is the case where pathway C is likely. In 1975, Martin and Webster published a paper dealing with the in vitro translation of f2 RNA (115). Th is work was done twenty years ago but is still quoted even now (116) due to the fact that this is the only paper which demonstrated convincingly that the 30s subunit may under certain conditions remain transiently on mRNA. The paper is quoted as suggestive evidence that the 30s subunits may not leave mRNA and be used for translational coupling (pathway D). Martin and Webster isolated the complex of phage f2 RNA and the bound 70s ribosomes at the initiation site of the coat cistron which is translated efficiently in vitro. Other cistrons of this RNA, such as the polymerase and lysis proteins, are not highly expressed in this system. By kinetic analysis of the change of the 70s ribosome-f2 RNA complex, 30s subunit-f2 RNA complex, free f2 RNA and ribosomal subunits, they concluded that the termination of translation resulted in the release of ribosomes as subunits. They observed the transient appearance of the 30s subunit-f2 RNA complex. They concluded that SOS subunits were released first at the termination codon. Furthermore, they added an excess of SOS subunits together with initiation factors and showed that the conversion of the 7OS-f2 RNA complex into the 3OS-f2 RNA complex was delayed. These results were interpreted to suggest that when the SOS subunits were added exogenously, the released 30s subunits bound them at the next 3’ distal initiation site to form the new initiation complex of the 70s ribosome. The authors concluded that the 30s subunits leave RNA and bind to the next cistron. They stated, “This is most likely dependent on the tertiary structure of the region around the

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intercistronic region making the next initiation site readily available to the just released 30s ribosomal subunit containing IF3.” Their experimental data suggest pathway C in this system. It should be noted that they also provided an example of pathway A. In an effort to show that the possible presence of initiation factors in their system was not responsible for the appearance of ribosomal subunits upon release from mRNA, they isolated polysomes and studied their conversion to single ribosomes in their system. The results supported their expectation, i.e., the absence of any appreciable level of initiation factor activity in their system, because they saw conversion of polysomes to only 70s ribosomes. This is clearly the case of pathway A as we usually observe in our model system as discussed in Section I. Only when exogenously added initiation factors were present could they observe the dissociation of the 70s single ribosomes into their subunits due to the subunit dissociating activity of IF3. Contradicting the possibility of pathway G, ribosomes were shown to be released at the end of the coat cistron of RNA phage MS2 by Grubman and Nakada (117). They concluded, “the ribosomes which have completed the synthesis of coat protein dissociate from MS2 RNA. Translation of the 3’ adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.” In this experiment, when the reaction mixture was diluted after the coat protein synthesis was started, no MS2 polymerase was synthesized unless new ribosomes were added to the reaction mixture. This was interpreted to mean that the ribosomes which finished translating the coat cistron could not be used to initiate the downstream polymerase gene before their release from mRNA. If the ribosomes translating the coat cistron moved to the polymerase cistron without leaving the mRNA, dilution should not have influenced the translation of the downstream cistron because ribosomes were already on the mRNA. 5. Translational Coupling in the trp Operon Oppenheim and Yanofsky (98) studied the enzyme activities of the tryptophan (trp) synthesizing system coded for by the trp operon in which the first gene (coding for anthranilate synthetase component I), trpE, carried nonsense mutations. They collected several lines of evidence which indicated that efficient translation of the downstream cistron (coding for anthranilate synthetase component II), trpD, required translation of the distal portion of trpE. In other words, trpD translation is coupled to translation of trpE. Thus, nonsense mutations in trpE were 10 times more polar on trpD than on other genes of the

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operon located further downstream. Furthermore, rho-defective strains did not alleviate the polar effect of the amber mutation, but nonsense suppressors did. This means that trpE-trpD mRNA was produced, but ribosomes did not translate the trpD cistron of the message unless they first translated through trpE. In a similar manner, Das and Yanofsky (118) as well as Aksoy et al. (99) established that translation of trpA, the last gene in the trp operon (coding for tryptophan synthetase a-subunit) is coupled to that of trpB, which precedes A in the trp operon (coding for the synthetase P-subunit). The evidence includes showing the polar effect of a frameshift mutation in trpB on the expression of trpA-ZacZ fusion protein but less polarity on the ZacA gene further downstream. Evidence was presented that this coupling has no bearing on transcription (99). Das and Yanofsky considered two models for the possible mechanism of the observed coupling. They were both based on the assumption that de ~OZIOinitiation of the trpD and trpA translation by free ribosomes is somehow inhibited. In fact, a weak hairpin structure exists in the region. The ribosome binding to the initiation site of the downstream cistron could be hindered by this structure. 1) In the absence of trpE or trpB translation, the trp mRNA exists in a form that excludes efficient initiation of the trpD or trpA by exogenous ribosomes from the free ribosome pool, and this barrier to initiation is removed by ribosomes translating the distal portion of trpE or trpB, respectively. This model belongs to either pathway A or B. 2) The ribosome or its component, which translates trpE or trpB also translates trpD or trpA, respectively. This model is in the scope of pathways C through F. In support of the second possibility, they note that the ends of trpE and trpB overlap with the start of trpD and trpA, respectively, in the sequence UGAUG. Referring to the observation of Martin and Webster (115) on the transient appearance of a 30S-mRNA complex, Das and Yanofsky also noted the possibility that the 30s subunit, which had terminated the upstream cistron, could immediately initiate the downstream cistron translation, “...since it is already bound at the downstream initiation site.” Testing the validity of our hypothesis that RRF is involved in this system is relatively simple with the use of tsfrr. In addition, it is possible to determine which one of their two hypotheses is correct. If their second hypothesis is correct, at the nonpermissive temperature of tsRRF, we would expect the ratio trpE/trpD and trpB/trpA to go up. This is because ribosomes would stay on mRNA and translate from the codon 3’ next to the termination codon, and would not synthesize

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D or A (45). Since there is residual protein synthesis after the temperature shift-up in E. co& tsfrr, this could be measurable. In addition, at the nonpermissive temperature of tsfrr, instead of trpD and trpA, we should observe a new protein with the NHz-terminal sequence Trp. Leu for trpD and Trp*Asn for trpA, respectively. This is because the . ..UGAUGGCUGAC... and intercistronic sequence is . . .UGAUGGAACGC. . . for trpE/trpD and trpB/trpA, respectively (229). UGG.CUG and UGG*AAC correspond to Trp*Leu and Trp* Asn, respectively. If their first mechanism is correct, we expect no change in the trpE/trpD and trpB/trpA ratio upon temperature shift-up provided that the ribosomes reading trpD or trpA out of frame do not interfere with correct reading by exogenous ribosomes. If the interference occurs, we expect the ratios trpE/trpD and trpB/trpA to increase upon temperature shift-up. However, the ratio change would be more dramatic with the second hypothesis than with the first. 6. Translational Coupling between galT and galK Schiimperli et al. (100) studied the possibility that the translation of the galactokinase (gaZK) cistron is coupled to the translation of the preceding galactotransferase cistron (galT) in the galactose operon of E. coli. In the wild-type operon, galT translational stop codon is separated by three nucleotides from the initiation codon of gaZK. The intercistronic nucleotide sequence is . ..UAA*GAA.AUG*AGU*CUG . . . . They constructed a set of plasmids with frameshift mutations or nonsense codons in gaZT, resulting in translational stops before, within and behind the galK initiation site. By measuring galK enzyme activity, galactokinase protein (in SDS-PAGE) and gaZK mRNA, they established the coupled expression which occurred at the level of translation (polar effect on the gene product but virtually no effect on the transcription of the message). They could see that the efficiency of translation of galK was inversely related to the distance between the translational termination site and the initiation site (in either direction). For the mechanism of this translational coupling, they considered three possibilities: a) terminating ribosomes in the upstream cistron melt out the secondary mRNA structures which possibly interfere with the initiation of translation of the downstream cistron by exogenous ribosomes (pathway A or B); b) terminating ribosomes from the upstream cistron reinitiate at gaZK without being released from the mRNA (pathway D or G); and c) terminating ribosomes are released and lead to an increase in local concentration of ribosomal subunits and

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reinitiate the downstream cistron (pathway C or F). They omit the first possibility (a) on the basis that, in one of their constructs (terminating 98 bases downstream of the guZK initiation site), the ribosomes had the capability to melt inhibitory mRNA structures during their translation of the upstream cistron, but translation of g&K cistron remained low. Involvement of RRF in this system can be tested in an experiment in which one of their plasmids (pDS44 (100)) will be used as template in in vitro transcription-translation assay with or without RRF. In the assay, galactokinase production will be monitored. Plasmid pDS44 directs a nearly wild-type level of galactokinase production. The gaZT-g&K junction in this plasmid is such that the last two nucleotides of the initiation codon for the galactokinase gene is the first two nucleotides of the termination codon for galactotransferase gene (in the AUGA sequence). Lack of galactokinase synthesis would be expected in the absence of RRF because the unreleased ribosomes would resume translation at the first available codon behind the guZT termination codon (45), which in pDS44 is out of the frame for guZK. This translation would end quickly because the intercistronic nucleotide sequence . . .AUGAGUCUGAAAA... (100, 120) dictates that the next codon from UGA correspond to Val (GUC) followed by the new termination codon UGA (100). In the absence of RRF, translation of this mRNA would resume again and produce a polypeptide having Lys as the NH*-terminal because the next codon AAA codes for Lys. 7. Translational Coupling in Ribosomul Protein Synthesis 1) S12 and S7 coupling (the str operon) The str operon consists of four genes: rpsL (r-protein S12), rpsG (rprotein S7), &A (elongation factor G), and t&A (elongation factor Tu). The S7 protein acts as a feed-back repressor on its own production, the synthesis of EFG and probably EFTu as well as S12 (121). It has been established that the major portion of S7 synthesis occurs via coupled translation to the upstream S12 translation (122). In a series of elegant experiments, Nomura’s group showed that a loop structure of the intercistronic region is important for the coupled translation of S7 (102). Almost every mutation in the stem structure lowered the coupling efficiency with few exceptions. In fact, a single base substitution in the base-paired region abolished 90% of the coupled translation of S12 and S7 by loosening the association between base paired regions locally. They suggest that the unusually large distance between the stop site in the upstream gene and the site of initiation does not fit well with the scanning model, pathway D.

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We believe that this system is consistent with either pathway C or F. The role of RRF can be tested in this system also. We propose that ribosomes skip the intercistronic loop. Such ribosomal skip is achievable when ribosomes are released from mRNA at the upstream termination codon. Ribosomes terminate the S12 synthesis and then the termination complex is broken down by RRF and EFG. The ribosomes from the broken down complex may not have to move far if the stem structure of the intercistronic sequence brings the initiation codon of S7 right next to the termination codon of S12 (UAA), which overlaps with the A of the AUG codon because of the loop which is skipped by ribosomes. This can be either pathway C or F. We postulate that the lack of RRF in this system may lead to the following consequences. After releasing nascent S12, the ribosome resumes translation on the S7 cistron but in +I frame and a new polypepSince the intercistronic nucleotide sequence is tide emerges. . ..UUAAUGGUUC-(84 nucleotides)-AUUUCCA UGCCACGT..., the NH*-terminal of this new +I frame-shifted protein will be Cys. His.... Note that, in our model, A of UAA is shared with A of AUG because of the ribosome “skip” of the 96 nucleotide loop. Thus, ribosomes would read UGC. CAC as Cys. His and produce + 1 frame protein. Since coupled S7 translation would be diminished, the S12/S7 ratio would go up in the absence of RRF. Alternatively, lack of RRF may allow ribosomes at the S12 termination codon to proceed to the next neighboring codon and continue translation of the intercistronic sequence. In this case, ribosomes must melt the stem loop structure. The NH*-terminus of the resulting peptide will be Trp. Phe, corresponding to UGG* UUC of the first six nucleotides of the intercistronic sequence. During this process, the initiation codon AUG of S7 cistron may or may not become available to the exogenous ribosomes. If it does, S7 synthesis may still go on. Due to the fact that the number of bases in the intercistronic region is 96 (multiple of three), it is also possible that S7 with extra amino acids at the NHZ-terminus may be produced. 2) Lll-Ll coupling Yates and Nomura (101) indicated that in the Ll 1-Ll ribosomal protein (r-protein) operon “sequential translation” (translational coupling) of the two cistrons is the mechanism by which the two r-proteins are produced in equimolar quantities. They based their statement on their in vitro finding that Ll (the distal cistron in the operon) inhibited the synthesis of itself and Lll. In a subsequent paper, Baughman and Nomura (123) supported this conclusion.

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Their model to explain this coupled translation is that the ribosome (or more likely its 30s subunit, referring to Martin and Webster) which finished the translation of the Ll 1 cistron is used for translation of Ll. Although this is consistent with pathways C and F, they appear to prefer pathway C. We can again test the validity of our model in this case with tsfrr. Since the intercistronic region contains 3 nucleotides, removal of RRF will not cause out-of-frame reading of Ll. Therefore, no appreciable change will be observed in the quantity of Ll VS. Lll. However, Ll produced in the absence of RRF (at the nonpermissive temperature of tsfrr) would have one extra NHz-terminal amino acid Glu (coded for by GAA) according to the intercistronic nucleotide sequence between Ll and Lll ,- UAA*GAA*AUG-* 8. Reinitiation of Translation in the lac Repressor RNA Pathways C, D, and F appear to have problems in a system where there are considerable distances between the upstream termination codon and the reinitiation site. One such example is found in the early region of the E. cob Zac repressor RNA (73). In this system translation reinitiation events triggered by nonsense codons occur over a long distance (approximately 90 nucleotides). However, close examination of this situation revealed that either pathway C, D or F is also applicable to this case. The reinitiation in this case is accomplished by two “bridging” mechanisms. First, the ribosome translates an open reading frame in between the upstream termination codon and the downstream restart site. The ribosomes which completed the translation of this open reading frame do not have to move far to restart again. Second, there exists a possible secondary structure which shortens the distance between the termination codon and the site of reinitiation. With the use of one of the constructs of Matteson et al. (73), we carried out a preliminary test for the validity of our model. We were able to demonstrate that removal of RRF in viva induces synthesis of a new protein which is not made in the presence of RRF. This result supports our in vitro experiment (45) and the model derived from this in vitro experiment as discussed in Section I Subsection 16, and is consistent with either pathway C or F. We thank Dr. D. A. Steege for the generous gift of her plasmids. 9. Bacteriophage Lysis Protein Synthesis Adhin and van Duin (116) suggest that terminated but unreleased ribosomes (probably the 30s subunit) reach the neighboring initiation

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codon by lateral diffusion along mRNA. This is pathway D. This hypothesis, called the scanning model for translational initiation, is based on their studies using RNA bacteriophage fr and MS2. These bacteriophages contain four cistrons, maturation, coat, lysis and replicase in the order from 5’ to 3’. The lysis protein cistron is peculiar in that its initiation site is within the coding region of the coat cistron and the termination site is within the coding region of the downstream replicase cistron (see the structure of the closely related R17 phage in Fig. 13). Translation of the lysis cistron is strictly dependent on the translation of the coat cistron. The model proposes that “30s subunits which were not released at the termination codon of the coat cistron will start scanning up and down along the phage RNA until they catch the initiation site of the lysis protein which is 35 nucleotides (phage fr) or 47 nucleotides (MS2) upstream of the coat cistron termination codon.” If the coat protein termination occurs upstream or downstream from two competent start sites for the lysis protein (one was created artificially), ribosomes will start preferentially from the nearest start site. The scanning model also explains a separate finding from the same laboratory by Schmidt et al. (124) with the MS2 RNA phage. The two dimensional structure of MS2 RNA near the initiation codon of the lysis cistron and the termination codon of the coat cistron can be visualized as a large stem loop structure. The results of mutational studies within this loop suggested that the 30s subunits scan through this loop. This system can be adapted to prove or disprove our hypothesis that RRF plays a crucial role in this translational coupling system. Since their system is in viuo, tsjrr can be adapted to their system. They used a series of plasmids containing the fr phage information downstream from the PL promoter in plasmid pPLa2411 (96). The fr genes are induced by raising the culture temperature to 42°C because the function of the promoter is regulated by the temperature sensitive repressor. To avoid complications with tsjrr (42°C is the nonpermissive temperature), the PL promoter has to be replaced with another inducible promoter such as the Zac promoter. The wild-type fr plasmid pFRl.0, containing the wild-type fr genes of the coat and lysis proteins (96) along with the Zac promoter, will be placed in E. coli with tsjrr. Activation of the pFRl.0 Zac promoter will lead to lysis at 32°C but not at 42°C. This is because the termination complex at the termination codon of the coat cistron will not be broken down at 42°C and therefore “scanning 30s subunits”

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will not be produced for the translation initiation of the lysis protein. This prediction is based on the assumption that lysis is triggered by a relatively small amount of the lysis protein but not dependent on active cell growth because E. coli with tsfrr stops growing at 42°C. Another assumption is that, at 42”C, sufficient residual Zac inducer protein can be synthesized without RRF. At 42”C, the Western blot analysis will reveal the presence of the coat protein predominantly, while the lysis protein synthesis will be reduced. Instead, the ribosomes will be making a new protein which is unrelated to the lysis protein because the translation will be out of frame of the lysis protein. This protein will have the NHz-terminal sequence Glu. Trp . Arg because the nucleotide sequence near the termination codon of the coat protein is . ..UAC* UAA* GAA* ACC * CGU.... GAA, ACC, and CGU code for Glu, Thr and Arg, respectively. It should be mentioned that the above expectations are based on the assumption that the 30s subunits of ribosomes which read the coat cistron will initiate the lysis cistron (pathway D). If the ribosomes translating the coat cistron leave the mRNA but make the lysis cistron available to the exogenous ribosomes (pathway A or B), we may not observe a reduction of lysis gene translation provided that the synthesis of new out-of-frame protein on the lysis cistron does not interfere with the lysis protein synthesis. In any case, the emergence of the new outof-frame protein should still be observed upon the temperature shiftUP. 10. Translational Coupling between the atpH and atpA Genes of the atp Operon The proton-translocation ATP synthetase of E. coli is composed of eight different subunits with a stoichiometry of the subunits a:c:b:d:a:y:@ (1:10:2:1:3:1:3:1) (125). Determinants of the subunits are organized into one operon (126) located at 84 min on the E. coli chromosome. Translational coupling has been demonstrated for several gene pairs in the operon (127-132) and is thought to be the main means of regulating expression of the subunits for the required stoichiomen-y. For example, the atpH (coding for the d subunit) and atpA (coding for the a subunit) are coupled. Rex et al. (133) established that a secondary mRNA structure sequesters the initiation region (the ShineDalgamo sequence and the initiation codon) of the downstream atpA cistron preventing de no~lo initiation and independent translation of

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atpA. However, once a ribosome which had translated the upstream atpH cistron initiates at the atpA initiation region and translates it, the inhibitory structure rearranges and becomes less inhibitory for the initiation at the atpA initiation site by free ribosomes. Hence, this structure works like a “gating device.” Reinitiation at the atpA initiation site by ribosomes coming from the upstream cistron is essential to turn this structure on. This requirement was proven by the use of mRNA whose Shine-Dalgarno sequences were altered into anti-Shine-Dalgarno sequences (ASD) and the employment of specialized ribosomes capable of recognizing ASD (134) by appropriate changes at the 3’ end of their 16s rRNA. Wildtype E. coli does not recognize ASD while E. coli which has the engineered 30s subunits in addition to wild-type 30s subunits can. Neither of the two constructs, the one with ASD in the atpA initiation region but wild type Shine-Dalgarno sequence in the atpH initiation region (construct I), or the one with ASD in the initiation regions of both genes (construct II) expressed atpA in E. coli with the wild-type ribosome. This indicated that, for the expression of atpA, the very ribosomes which had finished the translation of atpH have to reach the initiation site of atpA. The fact that construct I did not give atpA is consistent with pathways C through G. The same ribosomes or their subunits which translated the atpH cistron are responsible for translating atpA cistron. This was further confirmed by the second set of experiments. When the constructs were placed in E. coli with the engineered ribosomes in addition to the wild-type ribosomes, the double ASD construct (construct II) expressed atpA, but the one with ASD in front of the downstream cistron and SD at the front of atpH (construct I) remained “silent”. This indicated that translation at the upstream atpH cistron by wild-type ribosomes per se is not enough to “open the gate” for initiation at the atpA initiation region by the engineered ribosomes. The ribosome which performs this task must be the one which completed the translation of the upstream cistron, and it then binds to the initiation site of the downstream cistron. The above constructs could also be useful in proving or disproving our hypothesis that RRF exerts its effect on the termination complex at the end of the first cistron before coupling occurs. Namely, the SD-ASD construct (construct I) will be placed in E. coli with tsfrr and with no engineered ribosomes. The question can be asked whether the absence of RRF causes the expression of the A gene in this construct. We expect the answer is yes because the wild-type ribosomes

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bound to the initiation region and translating atpH would not be released from the mRNA at the termination codon of the cistron after tsRRF is inactivated. Instead, they would reinitiate translation at the next available codon downstream, translate the intercistronic region (12 base pairs long), and proceed further downstream to translate cistron atpA which, in this case, is in frame with the atpH cistron (134, 135). The intercistronic nucleotide sequence is . . .UCU . UAA * GGG . GAC*UGG.AGC*AUG*CAA.... As a consequence, the atpA product is expected to be modified by the addition of four amino acids (Gly*Asp*Trp*Ser) to its NH2 terminus coded for by the intercistronic sequence. The positive results of this experiment would strengthen the notion that the SD sequence of the A cistron is not necessary for translation of this cistron if the ribosomes are on the mRNA before they reach the initiation region. A negative control for this experiment is to use the construct which has ASD for both the A and H cistrons (construct II) in a tsfrr strain which has no engineered ribosomes that can use ASD.

III.

A POSSIBLE

ROLE

BY RRF IN mRNA DEGRADATION

Bacterial mRNAs are generally unstable and are quickly degraded by various endonucleases and exonucleases (12). The role of translation in mRNA stability is complicated and can have three different consequences depending on the cases ranging from positive correlation (136, 137), no relation (127, 138), and negative effect (translation may increase mRNA degradation (139)). However, there is more evidence, at least in prokaryotes, for the concept that ribosomes have protective effects on mRNA from degradation. Since the RRF function is to release ribosomes from mRNA, we briefly speculate on its role in mRNA degradation.

1.

Background and Our Model In our in vitro experiments, release of mRNA

from ribosomes by pure RRF and EFG did not cause any degradation of mRNA (see Section I). However, we have never tested the effect of RRF and EFG on the stability of polysomal mRNA in the presence of soluble mRNA-degrading enzymes. In this section we deal with a few examples in which RRF could play a crucial role in the degradation of mRNA and suggest experiments to prove or disprove such a role for RRF. Our model assumes that the presence of ribosomes on an mRNA inhibits its degradation. Consequently, the release of ribosomes from

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mRNA by RRF and EFG would facilitate mRNA degradation. Conversely, inactivation or removal of RRF would stabilize mRNA. The effects of antibiotics on mRNA stability support our assumption that ribosomes protect mRNA. Inhibitors of protein synthesis which stop ribosome movement and keep them on mRNA increase general mRNA stability. Thus, chloramphenicol (a peptide bond inhibitor (140)), tetracycline (an inhibitor of specific binding of aminoacyl tRNA (23)), fusidic acid (an inhibitor of translocation catalyzed by EFG (141,142)), and erythromycin (an inhibitor of translocation (243)) are all known to stabilize mRNA in E. coli. On the other hand, antibiotics which prevent ribosomes from binding to mRNA or promote release of ribosomes from mRNA, facilitate mRNA degradation. Thus, kasugamycin (an inhibitor of the initiation step (144)) and puromycin (an agent which removes the peptidyl group from the ribosome-bound peptidyl tRNA) increase degradation (145, 146). 2. Puromycin-induced Degradation of mRNA Of particular interest to us is the effect of puromycin. As described in Section I, puromycin treated polysomes are the substrate for RRF and EFG. Varmus et al. (145) pulse labeled mRNA in growing E. coli after P-galactosidase mRNA transcription was induced. After stopping synthesis of P-galactosidase mRNA by an antagonist to the /3-galactosidase inducer, decay of /3-galactosidase mRNA was examined by measuring labeled P-galactosidase mRNA hybridizable to P-galactosidase DNA. In the presence of puromycin, the half life of /3-galactosidase mRNA decreased from 2.3 min to 0.5 min. We hypothesize that puromycin facilitates mRNA degradation in viva by releasing ribosomes from mRNA through the action of RRF and EFG in viva, exposing the mRNA to degrading enzymes. The free mRNA, having lost the protection by ribosomes, is degraded by cellular ribonucleases. Our model can be tested relatively easily with the use of the temperature sensitive RRF. We expect that, at the nonpermissive temperature for tsfrr, puromycin would not destabilize /3-galactosidase mRNA because, without RRF, ribosomes would stay on mRNA and protect it. A negative control of this experiment is to examine the effect of kasugamycin at the nonpermissive temperature for tsfrr. Baummeister et al. (137) and Schneider et al. (147) measured the decay of P-galactosidase mRNA in the presence and absence of kasugamycin. They found that this antibiotic shortened the half life of mRNA by 50%. Since this effect is due to prevention of the formation of the initiation

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ET AL.

this

3. Possible Role of RRF in Releasing Ribosomes at the 3’ Physical End of mRNA The above mentioned kasugamycin experiment, however, may be complicated by the possibility that the absence of RRF may deplete free ribosomes by keeping all available ribosomes on mRNA. If so, kasugamycin would not decrease the mRNA stability in the absence of RRF because there would be no ribosome for kasugamycin to inhibit from binding to mRNA. This depends on whether or not release of ribosomes at the 3’ physical end of mRNA is dependent on RRF. If RRF is required for this process, removal of RRF would “freeze” all mRNA-bound ribosomes toward the end of mRNA. We have no information on this point but this question is answerable in vitro and in viva. In vitro, short synthetic mRNA without the termination codon can be used, and the question can be asked if releasing of ribosomes from that mRNA is dependent on RRF or not. In vivo, examination of the polysome pattern by regular physicochemical means such as sucrose density gradient centrifugation or by electron microscopy upon temperature shift-up may give information on this question. If release of ribosomes from the 3’ physical end of mRNA is dependent on RRF, we may observe an accumulation of ribosomes toward the 3’ end of mRNA at the nonpermissive temperature for tsRRF. This may protect mRNA from degradation from the 3’ end. 4. Nonsense-codon Induced Instability of mRNA Although the effect of antibiotics on mRNA stability is clear, the effect of premature termination on mRNA stability is not. In E. coli, the stability of bla #I-lactamase) mRNA is decreased or uninfluenced by insertion of a premature termination codon depending on the position of the insertion. Using the Sl nuclease digestion method which measures only intact P-lactamase mRfiA (the hybridization method measures broken fragments also), Nilsson et al. (148) showed that a premature termination codon placed at the 26th but not at the 56th position reduced the stability of mRNA. A similar experiment was carried out on ompA mRNA which is known for its unusually long half-life (15 min). When the premature termination codon was placed at the 29th position, it did not influence the stability of this mRNA. These systems can be used to examine the role of RRF in mRNA stability without use of puromycin. First, it would be of interest to

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examine the half-life of wild-type bla mRNA in the absence and the presence of RRF. It has been suggested that the short half-life of bla mRNA may be due to the fact that the speed of ribosome movement is faster than the speed of initiation of translation of this mRNA. Calculation by Nilsson et al. (148) showed that bla mRNA may often exist without ribosomes on it in E. coli. If so, prevention of the release of ribosomes from the normal termination codon at the nonpermissive temperature for tsfrr may result in an increase in stability. Examination of which portion of mRNA is preferentially protected may tell us whether or not RRF plays a role in pushing ribosomes off the 3’ physical end of mRNA (see the preceding subsection). Second, it will be worthwhile to study the mechanism of the increased mRNA instability due to the premature termination codon at the 26th position of this mRNA. Whether the increased instability is caused by release of ribosomes at this point, or by some other reason such as the structural change of mRNA due to introduction of the nonsense codon can be determined with the use of tsfrr. If the increased instability is abolished at the nonpermissive temperature for tsfrr, this would support the notion that the ribosome has to be released at this point for the mRNA to be degraded. Earlier, Morse and Yanofsky (149) also indicated that introduction of the nonsense codon in the tryptophan operon reduced the stability of the distal end of mRNA. Thus, their mutant 9802 (nonsense codon in trpE) and 9778 (nonsense codon in trpD) are polar mutants which caused faster degradation at the 3’ end of this mRNA. Our hypothesis predicts that in the absence of RRF (namely, at the nonpermissive temperature of tsfrr), the stability of trp mRNA of 9802 and 9778 would be restored. If so, it suggests that the reduced stability due to the nonsense mutation is not due to a possible structural change of mRNA caused by the nonsense mutation. 5. Nonsense-codon Induced Stability of mRNA In contrast to the examples above, a premature nonsense codon stabilizes mRNA in the following example. In studying the factors which influence mRNA half-life, Wagner et al. (250) found that an introduction of the nonsense codon at the 10th position increased the stability of P-galactosidase mRNA. They have measured the stabilization effect with two independent methods: a functional test of the /I-galactosidase and the actual measurement of the /%galactosidase mRNA in E. coli by the primer extension method. One explanation for their observation was that a translational stop causes the ribosome to pause and increases

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ribosomal protection from endoribonucleases at the 5’ end of the transcript. In fact, ribosomes have been shown to pause longer at termination codons than at regular internal codons by Bjijrnsson and Isaksson

(63). The outcome of RRF inactivation in this system may be one of the following: 1) if the stabilization effect is caused by the “long stay” of the ribosomes at the termination codon, the effect should not be influenced even if ribosomes continue to reinitiate at the codon next to the termination codon. Therefore, upon inactivation of RRF, the stability of the /3-galactosidase mRNA should not change. 2) If the nonsense codon stabilizes mRNA through structural change, loss of RRF should increase the stability further. This is because inactivation of RRF would allow ribosomes to stay longer on the mRNA. 3) Inactivation of RRF may abolish the “long stay” of ribosomes because translation from the next 3’ codon quickly moves ribosomes forward from the termination site. In this scenario, the inactivation of RRF may decrease the stability of this mRNA to that of wild-type mRNA.

IV.

DOES EUKARYOTIC

RRF EXIST?

We do not know whether a eukaryotic equivalent of RRF exists or not. Since there is no data on eukaryotic equivalent of RRF, all discussions in this section are strictly speculative. To emphasize this point, an eukaryotic equivalent of RRF is expressed as “eukaryotic RRF” to remind the reader of the possibility that it may not exist. Even if it exists, “eukaryotic RRF” may function differently from RRF.

1. Important

Differences between Eukaryotic and Prokaryotic Translation Apparatus with Respect to Possible RRF Function There are a number of differences between the translational machinery of eukaryotes and that of prokaryotes (reviewed in (151)). In addition to the well-known difference in the size and structure of ribosomes (80s ribosomes OS. 70s ribosomes) and in the mode of the initiation mechanism (met tRNA VS. fmet tRNA), important differences between these systems for “eukaryotic RRF” would be in the mRNA structure. Most eukaryotic mRNAs are functionally monocistronic (152, 153), while prokaryotic mRNAs are mostly polycistronic. This difference makes prokaryotic RRF essential because, without RRF, ribosomes would produce unwanted proteins and probably interfere with proper translation of the downstream cistron. On the other hand, loss of “eukaryotic RRF” function would not interfere with the trans-

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lation of the “downstream cistron,” because such a thing does not exist in most eukaryotic mRNA. These differences lead to an important difference in the mode of control of protein synthesis between eukaryotes and prokaryotes. For example, prokaryotes can synthesize equal amounts of two different proteins by coupling translation of two cistrons on the polycistronic mRNA. Instead of coupling the translation of mRNA, eukaryotes fuse these proteins and produce two different proteins by means of proteolysis of the fused proteins to ensure an equal ratio of two proteins. Therefore, efficient translational coupling, presumably through the action of RRF as we proposed in Section II, is not necessary for eukaryotes. There are a few exceptions to this rule especially with viral RNA. For example, Rous sarcoma virus has three open reading frames before the gag cistron (154) and the cauliflower mosaic virus mRNA has a “polycistronic” arrangement so that ribosomes have to reinitiate the downstream open reading frames. A similar situation exists with sv40 (255). Another important difference is that eukaryotic 40s subunits cannot initiate from an internal initiation signal, while prokaryotic 30s subunits can (reviewed in (157)). There are some exceptions to this rule also. Thus, Pelletier and Sonenberg (158) showed that an “internal ribosome entry site” (IRES, reviewed recently (156,159,160)) exists in poliovirus RNA which allows 40s subunits to enter at an internal site and initiate translation. A similar finding was reported by Jang et al. (161, 162) with encephalomyocarditis virus RNA. However, even these reports have been criticized, and the possibility remains that ribosomes may still bind to the end of the mRNA of these viruses and scan through the untranslated 5’ region (163). Given this inability of 40s subunits to enter mRNA internally, the translation of rare eukarymRNA will be hampered by the release of ribootic “polycistronic” somes from mRNA at the termination codon of the upstream cistron by presumed “eukaryotic RRF.” 2. “Eukaryotic RRF” May Not Release Ribosomes at Every Termination Codon The only cases where eukaryotic ribosomes can initiate internally in addition to IRES were documented with eukaryotic “polycistronic mRNA.” The internal initiation occurs when another upstream AUG codon exists with the strong “Kozak consensus” arrangement accompanied by a termination codon in-frame with this AUG codon. It is stated that the termination codon has to be upstream of the reinitiation

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site (reviewed in (164)). Th is would be pathway E as described in Section II. This means that there is no eukaryotic counterpart of the situation found in RNA phages, i.e., the initiation codon of lysis protein located in the upstream region of the coat cistron termination codon (see Section II Subsection 9). In other words, 40s subunits which finished translation of the upstream open reading frame cannot go back upstream and reinitiate at the second AUG within the upstream coding region. In fact, recent evidence suggests that the 40s subunit scanning cannot go upstream on mRNA (165). In other words, pathway D (Section II) does not exist in eukaryotes. This is very much against the concept that “eukaryotic RRF” releases 80s ribosomes or their subunits at the termination codon and their high local concentration enables released ribosomes to reinitiate at the nearby AUG codon (pathway C or F in Section II). If pathway C or F were correct in eukaryotes, one would not expect the diffusion to be unidirectional toward the 3’ end. We should observe some initiation upstream of the termination codon although the possible masking effect of the advancing ribosomes may diminish such initiation. The most convincing evidence that “eukaryotic RRF” may not release ribosomes at the termination codon in polycistronic mRNA comes from the experiment where the distance between the upstream termination codon and the downstream initiation codon was artificially changed. Using mRNA coding for preproinsulin, Kozak (166) asked how reinitiation is affected upon varying the position of the upstream termination codon relative to the second AUG. The efficiency of reinitiation progressively improved proportionally to the distance between the termination codon and the initiation codon. When the distance reached 79 nucleotides, the synthesis of preproinsulin was as efficient as if there were no upstream AUG codons. The increase of efficiency was more than ten fold when the distance was changed from zero (the termination codon overlapping with the initiation codon) to 80 nucleotides. This is in sharp contrast to the results of similar experiments on translational coupling in prokaryotes. In experiments involving E. coli and Bacillus subtilis operons, the efficiency dropped 4-50 fold, depending on the operon studied, as the distance between the termination codon and the downstream initiation codon increased from zero (overlap) to 48 nucleotides (103, 128, 167). These experiments indicate that the mechanism of translational

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coupling in prokaryotes is drastically different from that of eukaryotes. They speak against the possible release of eukaryotic ribosome at the upstream termination codon before the initiation at the downstream AUG. Since 40s subunits cannot initiate internally, ribosomes, once released, are not able to translate the downstream cistron. The possibility that local high concentration of “released” ribosomes may force them to enter mRNA internally became remote by this experiment because the longer the distance, the better the reinitiation. It is possible that the 40s subunits remain on mRNA at every eukaryotic termination codon (pathway E) including that of monocistronic mRNA. The 40s subunits may then start “scanning” the untranslated 3’ region of eukaryotic mRNA and fall off the 3’ physical end of the mRNA. Since 40s scanning at the 5’ end can be as long as 700 nucleotides, scanning the 3’ untranslated region (average 150 nucleotides, the longest is about 1,000 nucleotides (251)) may not be such a big task for the 40s subunits to perform. “Eukaryotic RRF” may then be needed only for dislodging 60s subunits from the termination complex as we discuss in Section II and Section IV Subsection 4. 3. Evidence for the Existence of “Eukaryotic RRF” There is circumstantial evidence that supports the existence of “eukaryotic RRF.” First, eukaryotes (plants) do possess a protein with an amino acid sequence extremely similar to that of RRF (76). As discussed in Section I, the function and cellular distribution of this protein is not known, but it could function like RRF because the sequence homology is striking. Second, single ribosomes or subunits accumulate at the expense of polysomes in eukaryotic cells in an environment that decreases the overall rate of protein synthesis. These data have been interpreted as suggestive evidence that, following completion of protein synthesis, ribosomes are released from polysomes in the form of either single ribosomes or subunits (61, 168-171). The third piece of evidence for “eukaryotic RRF” is that it has been shown in rat liver extract that puromycin treatment of polysomes does indeed release ribosomes from mRNA in much the same way as we observed with E. coli polysomes (272). In Hela cells, puromycin treatment increases SOS ribosomes (61) (the 80s ribosome is called the 74s ribosome in this paper because of its actual sedimentation velocity). This is almost a replica of the similar E. coli experiment (34). Although there is no evidence that puromycin-induced release of ribo-

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somes from mRNA in eukaryotes is enzymatic, similarity of these experiments to those E. coli experiments encourages us to think that “eukaryotic RRF” may exist.

4.

Possible Functions of “Eukaryotic RRF”

Given the likelihood that “eukaryotic RRF” does not release 80s ribosomes at every termination codon, we postulate that eukaryotic RRF may release 60s subunits from the termination complex. In Section II, we described the possibility that RRF may dissociate SOS subunits from the termination complex to accommodate pathways C and D. This possibility, as described above, is more realistic in eukaryotes. At the upstream termination codon of eukaryotic “polycistronic” mRNA, 80s ribosomes are believed to release 60s subunits, leaving 40s subunits on mRNA (pathway E, see Section I I Subsection 2). However, how this occurs is not known. No enzymes have been proposed to do this task. There is no description of eukaryotic initiation factors capable of doing this. In fact, as discussed in Section II Subsection 3, the prokaryotic initiation factors cannot dissociate 70s ribosomes into their subunits at the termination complex and keep the 30s subunit on the mRNA. This is because IF3 breaks down the complex of the 30s subunits, mRNA and tRNA into their components (6). If what we found in prokaryotes applies to eukaryotes, eukaryotic initiation factors should not be able to dissociate the 80s ribosome of the termination complex into their subunits. This is because the resulting complex of the 40s subunit and mRNA should then be readily broken down by eukaryotic initiation factors. It is then conceivable that the dissociation of the 60s subunits at the intercistronic termination codon from the 80s ribosome may actually be carried out by “eukaryotic RRF” and eEFI1 (eukaryotic equivalent of EFG). It should be recalled that prokaryotic 70s ribosomes freshly released from mRNA by EFG and RRF are much more prone to dissociate into subunits, suggesting that such a role for “eukaryotic RRF” is also possible. On the other hand, it is still possible that “eukaryotic RRF” releases 80s ribosomes at the termination codon of monocistronic mRNA. If so, we wonder how “eukaryotic RRF” knows when to release 40s subunits from mRNA and when not to. There may be a control mechanism of “eukaryotic RRF” activity in this sense. It may be of interest to investigate the nucleotide sequence surrounding the termination signal followed by the downstream initiation. The se-

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quence should then be compared with those of the termination codon of monocistronic mRNA to find out if there is any signal for 40s subunits not to leave from the former and to leave from the latter. It is also possible that “eukaryotic RRF” releases ribosomes at the physical 3’ end of eukaryotic mRNA. As described earlier, we do not know whether the release of prokaryotic ribosomes from the physical 3’ end of mRNA is dependent on RRF. It is possible that this function is carried out by RRF and EFG in both eukaryotes and prokaryotes. In addition, “eukaryotic RRF” may release ribosomes which have accidentally lost peptidyl tRNA during chain elongation. These ribosomes could be synthesizing unnecessary proteins missing the NHrterminal portion of the normal protein. “Eukaryotic RRF” may prevent synthesis of these kinds of wasteful proteins. There is also a possibility that “eukaryotic RRF” may be involved in translational error prevention as we observed with RRF in E. coli (Section I Subsection 18). Finally, as described earlier, it is possible that “eukaryotic RRF” may function as a transcription factor as suggested by such activity of E. coli RRF (Section I Subsection 18). The fact that a closely related eukaryotic protein, NLP, may be a nuclear protein suggests such a possibility.

SUMMARY

AND

FUTURE

DIRECTIONS

We showed that RRF and EFG together catalyze ribosomal recycling through the release of ribosomes from mRNA at the termination codon. In this process, tRNA is released from the ribosome. The process requires GTP. Absence of RRF leads to the synthesis of unwanted proteins, which have the NHz-terminal amino acids corresponding to the distal sequence of the termination codon. Three facts indicate the importance of RRF in bacteria. First, it is essential for E. coli growth. The null mutation is lethal. Second, inactivation of RRF in viva resulted in synthesis of proteins not observed.in the presence of RRF. Third, a gene similar tofrr (the gene encoding RRF) is present in other smaller prokaryotes with much fewer genes, suggesting that RRF is required for life’s fundamental processes. Future studies will elucidate the possible role of RRF in translation coupling and reinitiation. The possibility that RRF may be involved in the release of ribosomes from the physical 3’ end of mRNA, in translational error prevention, and in mRNA degradation should be explored. An understanding of the molecular interaction between EFG

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and RRF during the release of ribosomes at the termination codon should be achieved. As a first step toward this goal, the three dimensional structure of RRF should be elucidated by crystallography and nuclear magnetic resonance. It is fortunate that EFG has already been crystallized. RRF with ‘a molecular weight of 20,000, heat stability, high solubility, and high pK should be amenable to structural studies by X-ray crystallography and NMR. The ribosomal binding site for RRF should be localized at the molecular level. The active sites of RRF which interact with the ribosome, mRNA, tRNA, and EFG should also be determined. The molecular basis of ribosomal recycling in eukaryotes should be elucidated. The possible biological role of RRF-like protein in eukaryotes should be studied in connection with the role of RRF not only in protein synthesis but also in biological functions other than ribosoma1 recycling. That the eukaryotic ribosomal recycling mechanism may be fundamentally different from prokaryotes encourages further study of both eukaryotic and prokaryotic RRF because RRF could then be a target for antibacterial agents.

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