The requirements for specific sRNA binding by ribosomes

J. Mol. Biol. (1966) 18, 90-108

The Requirements for Specific sRNA Binding by Ribosomes C. G.

KURLAND

Department of Zoology, University of Wisconsin Madison, Wisconsin, U.S.A. (Received 10 October 1965, and in revised farm 15 February 1966) It is possible to prepare ribosomes from Escherichia coli which are virtually free from contamination by GTP, supernatant proteins, and active sRNA. Such preparations have been employed to determine the minimum requirements for the binding of a specific sRNA by the ribosomes. These are an appropriate mRNA to specify which sRNA shall be bound and ionic conditions that will permit the formation of a stable complex. There is no requirement for super· natant factors or GTP. The sRNA can be bound equally well in the amino. acylated form or in the deacylated form. It is concluded that the structural properties of the sRNA, mRNA and the ribosomes are sufficient per ee to determine the specificity of their interaction.

1. Introduction The ribosomes were the first elements of the protein synthetic apparatus to be identified (Borsook, Deasy, Haagen-Smit, Keighly & Lowy, 1950; Siekevitz & Zamecnik, 1951). In spite of this, their function in protein synthesis still -remains unclear. The discoveries of the function of mRNA (Brenner, J acob & Meselson, 1961; Gros et al., 1961) and sRNA (Crick, 1958; Hoagland, 1955; Hoagland, Zamecnik & Stephenson, 1957; Chapeville et al., 1962) have tended to obscure this problem. At present, the role of the ribosomes is thought to be the provision of nonspecific binding sites for mRNA and sRNA (Gilbert, 1963a,b,c; Cannon, Krug & Gilbert, 1963; Okamoto & Takanami, 1963; Takanami, 1963). However, the complexity of the ribosomal proteins (Waller & Harris, 1961; Waller, 1964) as well as the recent analysis of the mode of action of streptomycin (Davies, Gilbert & Gorini, 1964; Gorini & Kataja, 1964a,b) suggest greater complexity in ribosome function. Two particular difficulties have hampered the analysis of ribosome function. First, the ribosomes are structurally complex (Watson, 1963), which suggests that experimental conditions must be found that permit a clear separation of individual steps in protein synthesis. This requirement leads to the second problem: the difficulty of obtaining highly purified ribosomes which are active in protein synthesis. In the course of the present study, a new procedure was developed for the purification of ribosomes that are virtually free of supernatant contamination. These highly purified ribosomes have been employed to determine the minimum requirements for the specific binding of sRNA (Arlinghaus, Favelukes & Schweet, 1963; Kaji & Kaji, 1963; Spyrides, 1964). This reaction is of special interest because it is thought to be the only ribosome-mediated amino acid-specific step in peptide-bond synthesis. Whatever role the ribosomes have in this step must be essential to the specificity of protein synthesis. 90

REQUIREMENTS FOR sRNA BINDING

91

2. Materials and Methods (a) Bacteria

All of the experiments reported here were performed with fractions obtained from E. coli B. The bacteria were grown with forced aeration at 37°0 in a minimal medium which was enriched with casein amino acids (Kjeldgaard & Kurland, 1963). They were harvested in the later part of the logarithmic growth phase and stored as a frozen paste at -75°0. The pilot plant facility of the Biochemistry Department at the University of Wisconsin (supported by NIH grant FR-00214) was used for these preparations, and I am indebted to Dr J. Garver and Mr A. Olson for their help. (b) Ribosomes The present purification scheme employs ammonium sulfate in both a chemical fractionation and in differential centrifugation to obtain ribosomes. This use of ammonium sulfate is derived in part from the procedure of Elson (1958) and from the observation of Wood & Berg (1962) that high concentrations of ammonium sulfate do not inactivate the ribosomes. All of the steps were carried out at 0 to 2°0 unless otherwise stated. 25 g of frozen bacterial paste were slowly thawed in 15 ml. TSM (0'01 M-tris, 0·003 Msuccinic acid and 0·01 M-MgOI 2 , pH 8,0) which contained 10 fJ-g/ml. DNase. 1 ml. of lOX TSM (0'1 M·tris, 0·03 M-succinic acid, 0·1 M-MgS0 4 , pH 8'0) was added to the bacterial suspension, which was then disrupted in a French press at 15,000 to 20,000 lb.yin.", The extract was centrifuged for 30 min at 25,000 g. The resulting supernatant liquid was incubated with 30 fJ-g/ml. puromycin for 15 min at 32°0 in order to remove nascent protein. The crude extract was chilled and diluted to 100 ml. with TSM. The ribosomes were partially purified by 3 successive ammonium sulfate fractionations. To 100 ml, of the crude extract obtained in the previous step, 21 g of solid ammonium sulfate were added and the mixture was agitated for 3 min. To the supernatant fraction obtained from a 10-min centrifugation at 25,000 g an additional 21 g of ammonium sulfate were added as above. The mixture was centrifuged for 10 min at 25,000 g and the pellet obtained was dissolved in 100 m1. of TSM. The second ammonium sulfate fractionation was performed as described above, except that the fraction precipitating between 21 g and 38·5 g ammonium sulfate was taken. The third ammonium sulfate fractionation was performed exactly as described above, except that the fraction precipitating between 21 g and 35 g ammonium sulfate was taken. The final pellet was resuspended in TSM containing 0·6 M-ammonium sulfate and centrifuged for 3 hr at 150,000 g (Spinco no. 50 rotor at 50,000 rev.jmin), AIl of the super. natant liquid was discarded and the pellets were resuspended in the same buffered ammonium sulfate solution. The ribosomes were centrifuged again for 3 hr at 150,000 g and the supernatant liquid was discarded. Next, the ribosome pellets were suspended in TSM and dialyzed overnight against 3 successive 1-1. portions of TSM. Then the ribosomes were centrifuged for 2 hr at 150,000 g. The resulting ribosome pellets were resuspended in TSM, clarified by a 10-min centrifugation at 25,000 g and stored in small portions at -75°0 at a concentration of 500 A260 unita/ml. There was no observable change in the activity of these ribosome preparations after 1 month at -75°0. (c) Supernatant enzymes The soluble fraction II of Wood & Berg (1962) was used to aminoacylate sRNA as well as to catalyze polypeptide synthesis. All of the steps in this purification scheme were performed exactly as described by Wood & Berg (1962). Thawed frozen E. coli were disrupted by grinding with alumina and a crude extraet was prepared. The soluble fraction I was then prepared by an ammonium sulfate precipitation, dialysis and centrifugation. The final soluble fraction II (SF II) was obtained after a protamine sulfate precipitation and was stored at -75°0. (d) s RNA preparations A commercial sRNA preparation from E. coli B (General Biochemical Corp., Ohagrin Falls, Ohio) was used for these experiments. All of the radioactive aminoacyl-sRNA preparations were made as follows: The reaction mixture, in a volume of 5 ml., contained

92

C. G.KURLAND

the following: 0·01 M-MgCI2; 0·01 M-KCI; 0·01 M-ATP; 0·1 M-potassiwn cacodylate (pH 7'0); 0·01% bovine serum albumin, 0'05% 2-mercaptoethanol; 25 mg sRNA; 18 amino acids at 10 iLM (asparagine and glutamine omitted), 5 mg of protein from the SF II preparation and 10 iLM [14C]Phe (333 poc/pomole, New England Nuclear Corp., Boston, Mass.). This mixture was incubated for 30 min at 32°C and chilled. This incubation is sufficient to saturate the sRNA with amino acids. The sRNA was deproteinized by 2 phenol extractions and was then precipitated twice with 2 vol. of cold ethanol. The aminoacylated sRNA was dialyzed overnight against water; it was then freeze-dried and stored as a frozen concentrated solution at -20°C. The final [HC]Phe sRNA preparation had a specific activity of 3780 cts/min/A260 unit. Two sRNA preparations were aminoacylated with an hydrolysate of 14C·labeled yeast protein at a specific activity of 850 poo/mg protein (Schwarz Laboratories, Orangeburg, N.Y.) as described above. One of these preparations was aminoacylated with the HC-Iabeled amino acids and a 100-fold excess of unlabeled Phe (1 mM); the other preparation was acylated with undiluted HC-Iabeled amino acids. The Phe-quenohed sENA preparation had a specific activity of 4790 cts/min/A260 unit, and the unquenched one had a specific activity of 5100 cts/min/A260 unit. HC-terminal adenine-labeled sRNA ([HC]Ade sRNA) was obtained by resynthesizing the 3'-hydroxyl terminal trinucleotide sequence of partially degraded sRNA in the presence of [HC]ATP. The degraded sRNA was prepared by S. J. S. Hardy as described in Hardy & Kurland (1966, manuscript in preparation). The procedure of Preiss, Dieckmann & Berg (1961) was used to add the terminal adenosine, and the sRNA obtained had a. specific activity of 3620 cts/minfA 2BO unit. All of the sRNA preparations were deacylated, with the exception of those specifically designated as aminoacylated sRNA. This was done by a modification of the procedure of Sarin & Zamecnik (1964). The sRNA was incubated at room temperature for I hr in 1·8 Mtris (pH 8,5). Under these conditions, the Phe sRNA has a half-life of approximately 10 min. A deacylated sRNA preparation was partially purified for Phe-acceptor activity according to a modification of the procedure of Cherayil & Bock (1965). These fractionated samples were kindlysupplied by J. Chen. The Phe-acceptor sRNA was approximately threefold purified. The sRNA deficient in Phe-acceptor activity was obtained by pooling column fractions containing no appreciable Phe-acceptor activity. (e) Radioactive supernatant protein

A radioactive supernatant fraction was prepared to facilitate the analysis of ribosome purity in a reconstruction experiment. A culture of E. coli B was grown in a medium containing a HC-Iabeled hydrolysate of yeast protein (850 poo/mg protein). The bacteria were disrupted by grinding with alumina and a crude extract was prepared [Tisaieres, Watson, Schlessinger & Hollingworth, 1959). The ribosomes were removed from this extract by 2 successive centrifugations at 150,000 g for 3 hr. In each case the top half of the supernatant liquid was isolated. The final supernatant liquid contained 4440 cts/min/mg protein and was used as the source of radioactive supernatant protein. (f) Concentration and radioactivity measurements

Protein was measured by the procedure of Lowry, Rosebrough, Farr & Randall (1951) with muramidase (Worthington Corp., Freehold, New Jersey) employed as a standard. RNA concentrations were estimated spectrophotometrically and an extinction coefficient (Ei~~ml') of 227 was used (Kurland, 1960). Radioactivity was measured in a thin-window counter (Nuclear Chicago) with a background of 13 cte/min. In two of the experiments described here, a low-background counter (Nuclear Chicago) was used with a background of 1'5 cts/min. Both of these radioisotope counters operated with an efficiency of approximately 30% for HC-Iabeled material. The molar concentration of ribosomes was calculated, taking the molecular weight for the 70 s particle as 2'7 X lOB (T'issieres et al., 1959). It was assumed that the ribosomes were all 70 s particles; the concentration was estimated from spectrophotometric data plus the fact that these ribosomes are 67% RNA (see below). Thus, one A2BO unit of ribosomes corresponds to 44 f-Lg of RNA, 66 f-Lg of ribosomes or 24 l-'moles of 70 s particles.

REQUIREMENTS :FOR sRNA BINDING

93

(g) sRNA binding assay

The assay procedure of Nirenberg & Leder (1964) was employed in this study to measure the binding of a specific sRNA by the rihosome-mRNA complex. The routine assay mixture contained, in a volume of 50 fL!': 0·02 M-MgCI2 , 0·05 M-KCI and 0'1 M-tris-acetate (pH 7,2) (assay buffer). The standard assay mixture contained 2·5 A260 units of ribosomes, 3·0 A 260 units of sRNA and 50 JLg of a homopolymer, usually poly U (all homopolymers were obtained from the Miles Laboratories). This concentration of poly U represents a more than 10-fold excess over that required to promote maximum binding of Phe sRNA by the standard concentration of ribosomes. The mixtures were incubated at 37°C for the required time and then diluted with 3 ml. of cold assay buffer. The ribosomes were adsorbed to a Millipore filter (HA 0'45, Millipore Filter Corp., Waltham, Mass.) as described by Nirenberg & Leder (1964) and washed with two more 3·m!' portions of cold assay buffer. Then the filter was glued to a metal planchet. Since radioactive sRNA preparations were employed in this assay, the extent of binding was assayed by measuring the radioactivity associated with the ribosomes adsorbed to the filters. (h) Assay of poly phenylalanine synthesis Poly Phet synthesis was measured with 25 A 260 units of ribosomes in a O'25-m!. reaction mixture which contained: 8 mM-MgS0 4, 0·05 M-KCI, 0·3 mM-2-mercaptoethanol, each of 18 amino acids at 0·25 mx (asparagine and glutamine were omitted), 1 mM ATP, 0·25 mM GTP, 8 fLg poly U and 0·04 M·tris (pH 7,8). The [14C]Phe was added at a specific activity of 7 fLcfJLmole. Appropriate amounts of sRNA and SF II protein were added and the mixtures were incubated at 37°C. The reaction was stopped by the addition of 3 ml, of a solution containing 7% PCA and 1% casein amino acids. Then the mixture was boiled for 10 min and the hot acid-precipitable material was isolated as a pellet from a 10-min centrifugation at 8000 g. The pellet was dissolved in 1 ml. of 0·1 N-NaOH, and 3 ml. of the solution containing 7% PCA plus 1% casein amino acids were added. The resulting precipitate was isolated on a Millipore filter, washed twice more with the same acid solution and the radioactivity was counted.

3. Results (a) Stability of the system at 37°0

[140]Phe

The kinetics of sRNA binding to the ribosome-poly U complex are shown in Fig. 1. The maximum level of binding is observed within 15 minutes at 37°0; this level remains constant for at least one hour longer. An experiment was performed to determine whether or not the ribosomes are inactivated during the incubation at 37°0. If inactivation occurs, the plateau value for Phe sRNA binding after a 15-minute pre-incubation at 37°0 should be lower than the plateau value obtained with ribosomes that were not pre-incubated. This was not observed (Fig. 1); therefore, the ribosomes retain their activity for at least 30 minutes at 37°0. These results demonstrate that the ribosomes prepared by the present procedure retain their activity after prolonged incubations at 37°0 and that the sRNA-ribosome complex is stable at this temperature. (b) Specificity of binding

Table 1 summarizes the macromolecular requirements for Phe sRNA binding using the ribosome preparation described above; these are the ribosomes and a specific polymer to serve as mRNA. Poly U in this case cannot be replaced by poly A,

t Abbreviations used: poly Phe, poly phenylalanine; A 260 units, unit of material which in a I-m!. volume and light path of 1 em will have an optical density of 1 at 260 miL; poly A, polyadenylic acid; poly D, polyuridylic acid; poly C, polycytidylic acid; poly I, polyinosinio acid; PCA, perchloric acid: BSA, bovine serum albumin.

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FIG. 1. The kinetics of Phe sRNA binding at 37°0 were measured with 50·1'1. incubation mixtures that were assayed at times up to 40 min (-0-0-). The effect of pre·incubation at 37°C was determined with an identical series of reaction mixtures that were pre-incubated without sRNA for 15 min (-,6-,6-). Each reaction mixture contained 2·5 A2eo units of ribosomes, 2·5 A 260 units of [14C]Phe sRNA and 50 I'g of poly U in assay buffer.

TABLE

1

Requirements for Phe sRNA binding Incubation mixture

1. 2. 3. 4. 5. 6.

Complete Minus poly U Minus ribosomes Plus poly A; minus poly U Plus poly C; minus poly U Plus poly I; minus poly U

Ribosomes bound (ctsjmin)

[l4C]Phe sRNA (I'I-'ffioles)

2345

10·1 0·29 0·20 0·26 0·16 0·26

69 47 60 38 61

Each reaction mixture of 501'1. contained 1·1 A2eo units of [14C]Phe sRNA (3780 ctsjminjA2eO unit). The complete system contained ribosomes (2,5 A260 units) and poly U (10 f£g). Poly A, poly C and poly I were present in the same amounts as poly U. The reaction mixtures were incubated in assay buffer (0,1 M-tris, 0·02 M-MgCI2, 0·05 M-KCI, pH 7,2) for 30 min at 37°C. The assay was performed as described above.

poly C or poly I. These results are similar to those obtained by Nirenberg & Leder (1964). The specificity of aminoacyl-sRNA attachment to the ribosome-poly U complex was also determined. Two samples of sRNA were aminoacylated with a 14C-Iabeled hydrolysate of yeast protein, and one of these was aminoacylated in the presence of a 100-fold excess of unlabeled Phe. If the binding of sRNA to the ribosomepoly U complex is specific for Phe sRNA, then there should not be a significant amount of radioactivity bound to the ribosomes when the Phe-quenched [14C]aminoacyl-sRNA preparation is employed. The data in Fig. 2 show that this is the case. In order to demonstrate that this result is not due to a nonspecific effect, it is necessary to show that it is dependent on poly U. This was done by substituting poly A

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for poly U in the binding assay. It was observed t hat in the presence of poly A the amount of radioa cti ve mat erial bound to the ribo somes was t he same when either t he unquenched or Phe-quen ched sR NA preparations were used in the binding assay, 269 and 284 ct sjmin, respectively . Therefore, it is concluded t hat the binding of sR NA t o the ribo some-poly U complex under the present conditions is highly specific. (c) Stoichiometry of binding It is necessary for the expe riments describ ed below t o kno w at what concentrations sRNA or ribo somes are limiting the exte nt of t he specific binding reaction. Therefore, the dependence of t he reaction on concentration was measured. Varying amounts of p4C]Phe sR NA were added to a fixed am ount of ribosomes and the extent of binding was measur ed after 30 minutes (Fig. 3), which is twice as long as is necessary to observe the maximum binding at these concent rations. When saturating a mount s of sRNA (3,0 A260 units or more) are in cubated with 2·5 A260 units of ribos omes, 15 JLJLmoles of Phe sRNA are bound. Since thi s concentrat ion corresponds to 61 JLJLmoles of 70 s rib osomes in the reaction mixture, it follows that at most one of four rib osomes is capable of binding a Phe sRNA in the presence of excess poly U. Thi s calculation is base d on t he assumption t ha t one sRNA is bound per active ribosome. Another rib osome preparation which was used for the experiments described here was t wo-thirds as active as the ribosomes used in the experime nt described in Fig. 3. (d) Cofador requirements

The data presented in Fig. 4 show t hat the addition of ATP or GTP to the incubation mixtures has no significant effect on either t he initi al rate or the final amount of Phe sR NA bound by the ribosomes. In order to be confident that neither ATP

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FIG. 3. The concentration dependence of Phe sRNA binding was measured with amounts of sRNA up to 8·0 A 260 units/reaction mixture. The 50-1'1. mixtures contained 2·5 A 260 units of ribosomes and 50 JLg of poly U in assay buffer. They were incubated for 30 min at 37°0.

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FIG. 4. The effect of ATP and GTP on the binding of the Phe sRNA by the ribosome-poly U complex was measured in the presence of excess sRNA. Three series of 50-1'1. incubation mixtures were used: one had no addition (-0-0-), another contained 0·1 mM-ATP (-,6.-,6.-) and the last had 0·1 mM-GTP (-0-0-). Each reaction mixture contained 2·5 A260 units of ribosomes, 50 JLg of poly U and 5·0 A 260 units of [140]Phe sRNA. These mixtures were incubated in assay buffer at 37°0 for times up to 20 min.

REQUIREMENTS FOR sRNA BINDING TABLE

97

2

Contamination of ribosomes by GTP Stage of purification

1. 2. 3. 4. 5.

Crude extract (NH 4b S0 4 fraction First pellet Second pellet Third pellet

Total GTP (ctajmin)

Total GTP (mumoles]

Total nucleic acid (A260 units)

509,000 25,900 1200 32 8

1000 59 2·4 0·063 0·016

2120 755 632 608 432

1 p.mole of [14C]GTP (1 p.c/",mole) was added to 10 ml, of a crude bacterial extract obtained from 2·5 g of frozen E. coli paste. The ribosomes were purified according to the procedure described above and the contamination by [14C]GTP was measured at successive stages. Radioactivity was measured from dried samples containing in each case 50 A 260 units of nucleic acid and counted in a low-background counter to 960 ets (background 1·5 cts/min). The purification of ribosomes was carried out on a scale which was one- tenth of that normally employed.

nor GTP is required for the specific binding reaction, it is also necessary to demonstrate that the ribosomes are not saturated with such molecules. This was done by a reconstruction experiment with [140]GTP. The radioactive GTP was added to a cruue extract of the bacteria at a final concentration of 0·1 mM. The ribosomes were then purified and the amount of [140]GTP remaining with the ribosomes was measured at successive stages of the preparation (Table 2). The final 432 A260 units of ribosomes, which correspond to 28·6 mg or 10·6 mumoles of 70 s ribosomes, are contaminated by 0·016 mumole of GTP. This sets an upper limit of approximately one GTP molecule for every thousand 70 s ribosomes. Even in the improbable event that the [ 14 0]GTP was diluted by a tenfold excess of endogenous GTP, this is definitely not a saturating quantity of GTP. Therefore, it is concluded that there is no GTP requirement for the specific binding of an sRNA by the ribosomes. It is possible to determine in a similar fashion the contribution of supernatant factors to the specific attachment of an sRNA to the ribosomes. The contamination of the ribosomes with supernatant protein was measured in a reconstruction experiment using a radioactive supernatant fraction which had been isolated from bacteria grown on 140-labeled amino acids. The radioactive supernatant was added to an unlabeled bacterial extract. A small portion of this mixture was centrifuged for three hours at 150,000 g. The top 5 ml. of this supernatant fraction were analyzed and the specific activity of this portion (1830 cts/min/mg protein) was employed to determine the contamination by supernatant protein at various stages of the ribosome preparation. The data from this experiment are summarized in Table 3. Slightly more than 21 mg of ribosomes were obtained and these contained less than 60 fLg of supernatant protein or less than 0·3% contamination by weight. Assuming that this supernatant protein has an average molecular weight of 60,000, there would be only one such molecule for every eight 70 s ribosomes. The observation that these ribosomes are 67% RNA, as contrasted with the usual value of 60 to 63% (Tisaieres et al., 1959) is additional confirmation of the high purity obtained. It is concluded that the ribosomes 7

TABLE

3

Contamination of riboeomes by 8upernatant protein

Stage of purification

1. 2. 3. 4. 5.

Crude extract (~H4)2S04 fraction First pellet Second pellet Third pellet

Total nucleic acid (mg)

Total protein (mg)

Total supernatant protein (cts/min)

Total supernatant protein (mg)

73·4 30·2 21·5 17·7 14·3

105 32·1 II·8 8·61 7·01

106,000 29,000 1660 524 103

56·8 15·8 0·899 0·291 0·0566

Supernatant protein (% of total It~it ~ protein)

32 25 2·7

i-t 0·27

4 ml. of a radioactive supernatant fraction, which had been labeled with [14C]amino acids (see above), was added to 8 ml. of a bacterial extract prepared from 2·5 g of frozen E. coli paste. 2 ml. of this mixture were diluted in 10 ml. of TSM, centrifuged for 3 hr at 150,000 g and the top 5 ml. of supernatant was used to determine the specific activity of supernatant protein. This was 1830 cts/min/mg protein, which was used to calculate the amount of supernatant protein contaminating the ribosomes at successive stages of purification. Nuoleie acid was estimated from the absorbancy at 260 mp. and an extinction coefficient of 227 (El'~). Protein was estimated by the Lowry method. The radioactivity was measured from samples containing 10 A 260 units of nucleic acid, and 1 mg of BSit was added before the samples were precipitated with trichloroacetic acid. The samples, collected on a Millipore filter, were counted in a low-background (1,5 ctsjmin) isotope counter to 960 cts.

REQUIREMENTS FOR sRNA BINDING

99

prepared by the present technique are contaminated by an extremely small amount of supernatant protein and are not saturated with supernatant enzymes. When the same supernatant fraction (SF II) which is used for the aminoacylation of sRNA is added to a mixture of ribosomes, poly U, as well as ATP, GTP and appropriate salts, little or no poly Phe synthesis is observed (Fig. 5). However, if an amount of deacylated sRNA equivalent to one-tenth the amount of ribosomal RNA is added to this same series of incubation mixtures, the rate of poly Phe synthesis is greatly enhanced and becomes roughly proportional to the amount of supernatant protein added (Fig. 5). These results demonstrate the absolute requirement for supernatant fraction to catalyze polypeptide synthesis with these ribosomes. In addition, it can be concluded that there are insignificant quantities of active sRNA in both the ribosome and supernatant fractions.

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In order to determine the influence of supernatant factors on the binding of an sRNA by the ribosomes, it is necessary to find conditions which permit this reaction but prevent concomitant peptide-bond formation. It has been found that the conditions used to assay the binding reaction are well suited for such experiments, because of the high Mg 2 + concentration and the absence of GTP. Thus, there is a greater than tenfold reduction in the rate of poly Phe synthesis when the Mg2+

100

O. G. KURLAND

concentration is raised from its optimum of 8 mx to that used in the binding mixtures. Similarly, there is no detectable poly Phe synthesis in the absence of ATP and GTP: thus 94 cts/min were incorporated when ATP and GTP were omitted, as compared to a blank value of 72 cts/min and 3260 cts/min incorporated by the complete system in 15 minutes. The data in Table 4 show that the addition of supernatant fraction has no significant effect on the capacity of the ribosomes to bind Phe sRNA. This experiment was performed by adding amounts of supernatant fraction, which when normalized to the quantity of ribosomes present, are identical to those employed in the experiment of Fig. 5. The 8% increase in Phe sRNA binding is attributable to nonspecific adsorption resulting from the increase in amount of protein present in the incubation mixture as well as to a limited amount of peptide-bond synthesis. It may be concluded from this experiment that the low efficiency of Phe sRNA binding by the ribosomes cannot be due to the loss of some supernatant factor during purification. TABLE

4

Effect of supernatant fraction on the extent of sRNA binding Added supernatant protein (JLg)

1. 2. 3. 4. 5.

None 10 20 30 50

Ribosome bound (ctsjmin)

2048 2064 2134 2112 2225

[ 14 0 )P he

sRNA (JLI-'mole)

8·82 8·86 9·12 9·06 9·54

Each incubation mixture in 50 JLl. contained 2·5 A260 units of ribosomes, 5·0 A260 units of sRNA, 50 JLg poly U and amounts of supernatant protein, which when normalized to the amount of ribosomes present, are identical to those employed in Fig. 5. The mixtures were incubated in assay buffer for 30 min at 37°0.

p 4 0 )P he

The previous experiment demonstrates that the ribosomes per se determine the extent of binding; however, the rate of binding might be stimulated by the presence of supernatant factors. This possibility was tested by measuring the initial rate of binding in the presence of the highest concentration of supernatant fraction used in the previous experiment. The data in Fig. 6 demonstrate that there is no effect of supernatant factors on the initial rate of Phe sRNA binding by the ribosomes. These results permit the conclusion that supernatant fraction is not required for the specific binding of an sRNA by the ribosomes. (e) Binding of deacylated sRNA

Isotope dilution experiments were performed with aminoacylated and deacylated sRNA to determine whether or not the aminoacylated sRNA was preferentially bound by the ribosomes. When an amount of [HC]Phe sRNA sufficient to saturate the ribosomes is added to an incubation mixture containing a twofold excess of unlabeled sRNA, it is found that both the aminoacylated and deacylated sRNA dilute the radioactive material bound to the ribosomes to the same extent (Fig. 7).

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FIG. 7. An isotope-dilution experiment was performed with unlabeled aminoaeylated sRNA and unlabeled deacylated sRNA. Each 50-1'1. reaction mixture contained, in assay buffer: 2·5 A 260 units of ribosomes, 50 /Lg of poly U and 3·0 A 260 units of p'C]Phe sRNA. One series of mixtures was incubated with no additions (-0-0-); So second series contained 6·0 A260 units of unlabeled aminoacyl-sRNA (- 6 - 6 -), and a third series contained 6·0 A 260 units of unlabeled deacylatedsRNA (-\7-\7-).

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C. a.KURLAND

This suggests that the ribosome-poly U complex cannot distinguish a Phe sRNA which is aminoacylated from one which is deacylated. There is, however, a serious objection to this experiment. Since the radioactive label being observed is only the amino acid, it is not possible to conclude unambiguously that the deacylated and aminoacylated sRNA are bound equally well. Thus, a transacylation reaction in which the radioactive label of a [14C]Phe sRNA bound to the ribosome is transferred to an unbound deacylated sRNA would result in a dilution of the [14C]Phe bound to the ribosomes. However, such a reaction could occur without there being equal competition for ribosomal binding sites between the aminoacylated and deacylated sRNA. For this reason experiments were performed with a deacylated sRNA preparation which was labeled at the 3' terminus with [14C]adenosine. The [14C]Ade sRNA is not specifically labeled, so that each class of sRNA molecules is labeled. This creates special problems for the binding assay. Some sRNA is always trapped by the filters in the assay and the ribosome-poly U complex enhances this effect by occluding the filter pores. This is a negligible effect when [14C]Phe sRNA is employed in these assays, but becomes a substantial effect with the [14C]Ade sRNA. This background of trapped radioactivity was estimated by measuring the amount of radioactive material adsorbed to the filters after a five-minute incubation at DoC. Such an estimate of the background is a maximum one. It is justified by the fact that the temperature dependence of the binding reaction is such that, in a fiveminute incubation at DoC, less than 5 % of the maximum level of [l4C]Phe sRNA will be bound. The binding of [14C]Ade sRNA is proportional to the amount of ribosomes present (Fig. 8) and its dependence on concentration is similar to that of [14C]Phe sRNA (Fig. 9). Therefore, it may be concluded that the binding observed with this [14C]Ade

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103

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FIG. 9. The [BC]Ade sRNA concentration dependence for binding to the ribosome-poly U complex was measured. Each 50-pI. reaction mixture contained 2·5 A 260 units of ribosomes and 50 p,g of poly U in assay buffer. Amounts of [14C]Ade sRNA up to 4·8 A 260 units/reaction mixture were added. The background of adsorbed radioactive material was measured after 5 min at O°C ( - 6 - 6 - ) and the binding of sRNA was measured after 20 min at 37°C (-0-0-). The uncorrected data are shown in (a), while the difference between the 37°C and O°C binding values are plotted in (b).

sRNA preparation is in no way different from that obtained with other sRNA preparations. The relative amounts of [BC]Ade sRNA and [14C]Phe sRNA bound by the ribosome-poly U complex can be estimated. When [14C]Phe sRNA (Phe specific activity 333 pc/pmole) is added in excess to ribosome-poly U complex, 3900 ctsjmin above background are bound. When excess [14C]Ade sRNA (adenine specific aotivity 10·1 /loc/pmole) is added to a parallel inoubation mixture, radioaotive material giving 170 cte/min above background is bound. This indicates that there are approximately 1-4 times as many [14C]Ade sRNA as [14C]Phe sRNA molecules bound by the ribosomes. Control experiments have shown that the specific activity of the Phe sRNA could be diluted by endogenous Phe in the aminoaoylation mixture to the extent of 20% at most. The specific activity of 10·1 /loCi/lomole is a maximum value, since it is not known whether there is unlabeled sRNA which can be bound in this preparation. The observed discrepancy between the amounts of [14C]Phe sRNA and [HCJAde sRNA bound by the ribosome-poly U complex can be accounted for by the presenoe of a small fraction of deacylated Phs-acceptor RNA in the [14C]Phe sRNA preparation. These corrections in any event could not lead to relatively more [14C]Phe sRNA binding than [14C]Ade sRNA binding. Therefore, the binding assay which employs [14C]Phe sRNA is not invalidated by limited peptide-bond formation. An isotope dilution experiment was performed with the [14C]Ade sRNA to determine whether or not deacylated sRNA is specifically bound and whether or not aminoacylated sRNA is preferentially bound by the ribosome-poly U complex. Excess radioactive sRNA was added to the incubation mixtures, and the dilution of ribosome-bound radioactive material by increasing amounts of unlabeled deacylated and unlabeled aminoacylated sRNA was measured. The interpretation of this experiment depends on the assumption that the specificity of sRNA binding is

C. G. KURLAND

104

reflected in the number of sRNA molecules which can be bound per A260 unit of sRNA. Thus if deacylated sRNA could be bound nonspecifically, the number of sRNA molecules which could be bound in a deacylated preparation would be much greater than that of the corresponding aminoacylated preparation. In this case the binding of [14C]Ade sRNA would be diluted much more efficiently by deacylated sRNA than by aminoacylated sRNA. Alternatively, it could be argued that a small residual fraction of aminoacylated [14C]Ade sRNA was responsible for all of the binding observed with this preparation. This is improbable, since the deacylation procedure employed in the preparation of this sRNA would leave less than 4% of the Phe sRNA intact. Furthermore, if a small amount of aminoacylated sRNA were responsible for all of the binding observed with the [14C]Ade sRNA, aminoacylated sRNA should dilute this binding of radioactive material much more efficiently than deacylated sRNA. The data in Fig. 10 show that unlabeled sRNA dilutes the binding of [14C]Ade sRNA to the ribosome-poly U complex equally well in the aminoacylated and deacylated form. Two conclusions may be drawn from this experiment. First, the ribosome-poly U complex cannot distinguish an aminoacylated sRNA from a deacylated sRNA under the present conditions. Second, the binding of deacylated sRNA is as specific as that for aminoacylated sRNA. The previous experiment suggests that only the relative number of Phe-aceeptor RNA molecules per A 260 unit of RNA, and not the degree of aminoacylation of this RNA, will determine the relative efficiency with which unlabeled sRNA will dilute

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FIG. 10. An isotope dilution experiment was performed with unlabeled deacylated sRNA, unlabeled aminoacylated.sRNA and [BC]Ade sRNA. Each 50-".1. reaction mixture contained 2·5 A 260 units of ribosomes, 50 ",g of poly U and 3·5 A 260 units of [14C]Ade sRNA in assay buffer. Two series of reaction mixtures: one with varying amounts of unlabeled deacylated sRNA (- 0 -0-), and the other with unlabeled aminoacylated-sRNA (-Ll.-Ll.-) were incubated for 30 min at 37°C. The binding values for [14C]Ade sRNA are plotted in (a) as a function of the amount of added unlabeled sRNA and in (b) as a function of the moles per cent of unlabeled sRNA. The dashed curves represent the theoretical curve expected if there is stoichiometric equivalence of the labeled and unlabeled sRNA.

REQUIRE MENT S FOR aR N A BINDING

105

the binding of radioactive sRNA to the ribosome-poly U complex. This was dir ectly tested in an isotope dilution experiment with partially purified deacylated sR NA (Fig . 11). The frac tion enri ched threefold for Phe-accept or activity diluted the binding of [14C]Phe sRNA three t imes more effect ively, on a weight basis, than did the unfractionated sRNA. In contrast , the fraction deficient in Phe-acceptor sR NA has only a slight effect on the binding of [14C]Phe sR NA. Th ese results demon strate that the binding of deacylated sRNA is specific. There is a small deviati on from the theoretical dilution cur ve in Fig. 10 and a greater one in the exp eriment described in Fig. II. The t heoretical curve is based on the assumption that the number of sRNA molecules which can be bound per A260 unit of RNA is the same for the labeled and unlab eled sRNA. However, control ,---.,.---,---,----,-r--.,.---,----,-----,,....------, 20 0 0

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FIG. 11. An isotope dilution experiment was p erformed with partially fraction ated, deacylat ed aRNA an d [HC]Phe aR NA. E ach 50· fLl. r ea ct ion mi xture contained 2·5 A280 u nit s of r ibosomes, 50 fLg of poly U and 3·3 A280 unite of [HC]Phe sRNA in assay buffer. One series of in cubati on m ix tures contained v arying a mounts of unfractionated deacyl ated sRNA (- 0- 0- ), t he sec ond ser ies of mixtures contained deacyla t ed sRNA whic h was deficient in Phe-acc ep tor activ it y (-0 -0 -) and the third seri es contained deacyl at ed sRNA which had been enriched for Pheacc eptor activit y ( -,6 - ,6 -). The bind ing values for [ 14 ClPhe sRNA are plo tted as a funct ion (a ) of t he amount of added unlabeled sRNA an d (b) of the mo les per cent of unlabeled sRNA.

experiments have shown that t his is not the case for different batches of sRNA. The labeled and unlabeled sRNA preparations used in the experiment described in Fig. 10 were from the sam e bat ch of sRNA, whereas the lab eled and unlabeled preparations used in the exp eriment described in Fig. 11 were from different batches. This would account for the larger deviation from the t heoret ical dilution cur ve seen in Fig. II. A small er contribution to such a deviation is made by the background of radioactive sRNA trapped in the filter, for which no correction has been made.

4. Discussion Th e specific binding of an sR NA molecule by a ri bosome- mR NA complex has been observed in vitro wit h bacterial and mammalian ribosomes (Arlinghaus et al., 1963; Kaji & Kaji, 1963; Spyrides, 1964; Nirenberg & Leder , 1964). H owever, the macromolecular requirements for t his reaction have not been clearly defined. The original experiments of K a ji & K aji (1963) employed an unfractionated bact erial extrac t from which the cell wall debris had been removed. Therefore, it is imp ossible to know whether or not the bindin g of nominally deacylated sRNA in this syste m

106

e.G. KURLAND

occurs without prior enzymic modification of the sRNA (e.g., aminoacylation). Subsequent work by Kaji & Kaji (1964), while more suggestive, did not rigorously demonstrate the equivalence of deacylated and aminoacylated sRNA in the binding reaction. Spyrides (1964) has pointed out that the ribosomes used in his studies were contaminated by an indeterminate amount of supernatant protein. This might mask a requirement for additional supernatant factors to promote the binding of sRNA. Arlinghaus et al. (1963; Arlinghaus, Shaeffer & Schweet, 1964) have observed that the binding of a specific sRNA by the ribosomes of the rabbit reticulocyte requires the addition of GTP and a supernatant enzyme. However, neither Spyrides (1964) nor Kaji & Kaji (1964) were able to demonstrate these requirements for the binding reaction by E. coli ribosomes. An analysis of the mechanism through which the ribosomes specifically bind an sRNA demands an unambiguous determination of the requirements for this reaction. In particular, it is necessary to know whether or not supernatant enzymes and an energy source are required. It is useful to use for such an investigation supernatant enzyme as well as ribosome preparations which meet certain minimum standards. The ribosomes should be obtainable free of significant contamination by supernatant protein, sRNA, small organic molecules such as GTP, and polymer-degrading enzymes. Similarly, a suitable supernatant preparation should not only require ribosomes for polypeptide synthesis, but must also be free of significant amounts of sRNA, amino acids, ATP, GTP and mRNA. The ribosomes used in the present study as well as the SF II supernatant fraction o(Wood & Berg (1962) meet these requirements. The present data show that ribosomes which do not contain significant quantities of GTP or supernatant protein are capable of binding a specific sRNA when provided with an appropriate mRNA. The addition of ATP, GTP or an active supernatant preparation to the assay mixtures has no significant influence on either the extent of sRNA binding or the rate at which it occurs. It is therefore concluded that there is no energy requirement or supernatant enzyme requirement for this reaction. The experiments described here do not explicitly rule out the involvement of a special class of non-ribosomal proteins. These would be proteins which are either not present in the radioactive supernatant fraction or proteins which are so tightly bound to the ribosomes that there is no exchange reaction with their radioactive counterparts during the reconstruction experiment. However, several considerations minimize concern about such hypothetical contaminants. First, the purity of these ribosomes is reflected not only in the small quantities of supernatant protein which apparently contaminate the preparation, but also in the observation that enzymes previously considered irreversibly bound to the ribosomes are removed in the purification (Hardy & Kurland, 1966, manuscript in preparation). Second, the fact that the addition of a highly active supernatant preparation has no effect on the ribosome activity makes it quite unlikely that some protein contaminant is necessary for all of the activity observed with the ribosome alone. Finally, if there are proteins irreversibly bound to the ribosomes and such proteins are required for ribosome function, then these should be called ribosomal proteins, at least until more sophisticated experimental criteria of what constitutes a ribosome are at hand. No stimulation of the rate of Phe sRNA binding by the ribosome-poly U complex was observed when active supernatant protein was added to the incubation mixture. These experiments were performed under conditions that do not permit peptidebond formation. Therefore, it is still possible that supernatant enzymes would

REQUIREMENTS FOR sRNA BINDING

107

stimulate the rate of sRNA binding under conditions of active peptide-bondformation. Indeed, Arlinghaus et al. (1963,1964) have observed that GTP and a supernatant enzyme stimulate the binding of sRNA to rabbit reticulocyte ribosomes. However, in the absence of a kinetic analysis of this phenomenon, it is not possible to decide whether or not this stimulation reflects a requirement for these cofactors in the binding reaction. For example, it is possible that GTP and supernatant enzyme are catalyzing the movement of previously bound sRNA from the initial binding site to a second site. In this way these cofactors would increase the number of available sites for the specific binding of sRNA but not participate at all in this primary event. The demonstration that the ribosome-poly U complex is unable to distinguish a Phe-acceptor RNA which is acylated from one which is deacylated is consistent with the adaptor hypothesis (Crick, 1958; Chapeville et al., 1962). This observation not only confirms the fact that the amino acid per se is not involved in the translation step, but it also implies that the ribose of the 3' terminus is not involved in the binding reaction. A problem is created by the equivalence of the deacylated and aminoacylated sRNA in the specific binding reaction. The propagation of the polypeptide chain on the ribosome is thought to proceed through the step-wise transfer of the growing chain to each incoming sRNA by the formation of the peptide bond (Gilbert, 1963c). The presence of a deacylated sRNA on the ribosome would therefore interrupt this process. There are two solutions to this dilemma: either the sRNA can be aminoacylated on the ribosome, or it can be displaced by an aminoaoylated sRNA. The next paper in this series will describe a specific exchange reaction between deacylated sRNA molecules bound to the ribosome-mRNA complex and unbound aminoaoylsRNA, which provides a solution to this problem. A multiplicity of sRNA binding sites has been postulated by Warner & Rich (1964) as well as by Wettstein & Noll (1965) for mammalian ribosomes. The present experiments do not confirm or exclude the existence of multiple sites for sRNA binding by E. coli ribosomes. Furthermore, the data presented here do not exclude the possibility that other kinds of sRNA binding sites with different requirements can be demonstrated under different conditions. The data presented in this paper show that the specific binding of sRNA by at least one of the binding sites of the ribosome-mRNA complex does not require ATP, GTP or supernatant enzyme. Furthermore, the sRNA can be bound at this site in either the deacylated or the aminoacylated form. Two conclusions can be drawn from these observations: first, this specific binding of sRNA is a thermodynamically spontaneous reaction; second, the primary structures of the sRNA, mRNA and the ribosome are sufficient per se to ensure the specificity of this reaction. I am grateful to J. Chen, S. J. S. Hardy and T. Likover for their help in preparing some of the material used in these experiments. Thanks are due to J. Adler and B. Weisblum for critically reading the manuscript. This work was supported by a grant from the National Institutes of Health of the United States Public Health Service. REFERENCES Arlinghaus, R., Favelukes, G. & Schweet, R. (1963). Biochem. Biophys. Res. Oomm. 4, 92. Arlinghaus, R., Shaeffer, J. & Schweet, R. (1964). Proc. Nat. Acad. s«, Wash. 51, 1291. Boorsook, H., Deasy, C. L., Haagen-Smit, A. J., Keighly, G. & Lowy, P. H. (1950). J. Biol. Ohem. 187, 839.

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Brenner, S., Jacob, F. & Meselson, M. (1961). Nature, 190, 576. Cannon, M., Krug, R. & Gilbert, W. (1963). J. Mol. Biol. 7, 360. Chapeville, F., Lipmann, F., von Ehrenstein, G., Weisblum,B.,Ray,Jr., W. J. & Benzer, S. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1086. Cherayil, J. D. & Bock, R. M. (1965). Biochemistry, 4, 1174. Crick, F. H. C. (1958). Soc. Exp. Biol. Symp. 12, 138. Davies, J., Gilbert, W. & Gorini, L. (1964). Proc. Nat. Acad. s«, Wash. 51, 883. Elson, D. (1958). Biochim. biophys. Acta, 27, 207. Gilbert, W. (1963a). J. Mol. Biol. 6,374. Gilbert, W. (1963b). J. Mol. Biol. 6, 389. Gilbert, W. (1963c). Oold Spr, Harb. Symp. Quant. Biol. 28, 287. Gorini, L. & Kataja, E. (1964a). Proc. Nat. Acad. Sci., Wash. 51, 487. Gorini, L. & Kataja, E. (1964b). Proc. Nat. Acad. Sci., Wash. 51, 955. Gros, F., Gilbert, W., Hiatt, H., Kurland, C. G., Risebrough, R. W. & Watson, J. D. (1961). Nature, 190, 581. Hoagland, M. B. (1955). Biochim. biophys. Acta, 16, 288. Hoagland, M. B., Zamecnik, P. C. & Stephenson, M. L. (1957). Biochim, biophys. Acta, 24,215. Kaji, A. & Kaji, H. (1963). Biochem, Biophys. Res. Oomm. 13, 186. Kaji, A. & Kaji, H. (1964). Proc, Nat. Acad. Sci., Wash. 52, 1541. Kjelgaard, N. O. & Kurland, C. G. (1963). J. Mol. Biol. 6, 341. Kurland, C. G. (1960). J. Mol. Biol. 2, 83. Lowry, O. H., Rosebrough, N. J., Farr, A. G. & Randall, R. J. (1951). J. Biol. Chem, 193,265. Nirenberg, M. & Leder, P. (1964). Science, 145, 1399. Okamoto, T. & Takanami, M. (1963). Biochim, biophys. Acta, 76, 266. Preiss, J., Dieckmann, M. & Berg, P. (1961).J. Biol. Ohem, 236,1748. Sarin, P. S. & Zamecnik, P. C. (1964). Biochim. biophys. Acta, 91, 653. Siekevitz, P. & Zamecnik, P. C. (1951). Fed. Proc. 10, 246. Spyrides, G. J. (1964). Proc, Nat. Acad. s«, Wash. 51, 1220. Takanami, M. (1963). Biochim. biophys. Acta, 72, 237. 'I'issieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. (1959). J. Mol. Biol. I, 221. Waller, J. P. (1964). J. Mol. Biol. 10, 319. Waller, J. P. & Harris, J. 1. (1961). Proc. Nat. Acad. Sci., Wash. 47, 18. Warner, J. R. & Rich, A. (1964). Proc. Nat. Acad. sa; Wash. 51, 1134. Watson, J. D. (1963). Science, 140, 17. Wettstein, F. O. & Noll, H. (1965). J. Mol. Biol. 11,35. Wood, W. B. & Berg, P. (1962). Proc, Nat. Acad. Sci., Wash. 48, 94.