Evidence for endonucleolytic attack in decay of lac messenger RNA in Escherichia coli

J. Mol. Biol. (1974) 83, 143-161

Evidence for Endonucleolytic Attack in Decay of lac Messenger RNA in Escherichia coli MARTIN BLuNDELLt AND DAVID KENNELL Department of Microbiology Washington University iWwo1 of Medicine Xt. Lo&s, MO. 63110$, U.X.A. (Received 10 May 1973, and in revised form 2 October 1973) The size distribution of decaying messenger RNA molecules from the lactose (Zac) operon of Escherichiu coli has been measured. A one-minute induction period was terminated by rifampicin, then a further period was allowed for completion of the Zuc mRNA transcription. All incomplete Zac mRNA molecules could then be identified as degradation products. RNA was labeled with [3H]uracil and purified by procedures which produced no significant cleavage. The purified RNA was centrifuged in sucrose gradients and then each fraction was hybridized with excess 480dlac DNA to determine the amounts of Iw message as a function of size. The size distributions observed at various times have been compared to those expected according to different mechanisms of degradation. These include a 5’ to 3’ exonucleolytio degradation as opposed to mechanisms in which the primary attack is endonucleolytic. The results clearly exclude an exclusive exonucleolytic degradation. Although the number and distribution of sites subject to endonucleolytic cleavage cannot be determined, the results are consistent with a model in which there are vulnerable sites at the start of the message specifying each polypeptide chain.

1. Introduction Messenger RNA instability can be observed by following either of two parameters: its loss of capacity to function as a template for protein synthesis (Kepes, 1963) or its (mass) disappearance from the cell (Morse et al., 19693; Schwartz et al., 1970). By both criteria the decay is exponential and commences very soon after the initiation of synthesis, although the two rates may differ (Schwartz et al., 1970; Kennel1 & Bicknell, 1973). In Escherichia cobi each message has a unique functional decay rate which can differ even from that of another on the same polycistronic mRNA molecule (Blundell et al., 1972). In t#hecase of the lac mRNA the temperature coefficients for decay of the first and last messages differ very markedly (Kennell & BickneU, 1973) and at 37°C the thiogalactoside transacetylase (last) messagedecays two to three times faster than the/3-galactosidase (first) message(Kepes, 1967 ; Blundell et al., 1972 ; Kennel1 & Simmons, 1972). These observations appear to exclude a mechanism whereby mRNA is inactivated and degraded exclusively by a 5’-exonuclease and suggest t Present address: Department of Biochemistry, Cambridge CB2 lQW, England. $ Address for reprint requests. 143

Cambridge University,

Tennis Court Road,

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that each message+ is inactivated by attack at one or a limited number of targets for that message, such as the ribosome loading site or an untranslated region just before its 5’-end (Blundcll et al., 1972). The model of a 5’ to 3’ “wave” of degradation was derived from studies of trp operon expression (Morikawa t Imamoto, 1969; Morse et al., 1969b); however, t,he fact t.hat deca.y commences as soon as the molecules are initiated means that the apparent direct.ion of decay need only reflect the 5’ to 3’ direction of synthesis. Thus, there is no direct evidence for either an exclusive 5’ to 3’ directional decay or for a decay that includes endonucleolytic attack. It is possible to distinguish exclusive 5’ to 3’ exonucleolytic degradation from various endonucleolytic models by measuring tho size distribution of the fragments of a decaying population of messenger RSA. To ensure that the observed distribution is meaningful, the RXA mu.& not be damaged by procedures used in extraction and fractionation. A suitable method is described. Our measurements follow the degrada.tion of a single well-characterized mRNA species; that transcribed from the lac operon of E. c&z’. The distribution of sizes is compared to t,hat predicted by various models of decay. While the results cannot define the endonucleolytic targets, t,hey clearly eliminate exclusive 5’ t.o 3’ exonuclcolytic decay as a mechanism for degradation and present direct evidence for internal cleavages during decay of this mRKA.

2. Materials and Methods (a) Bacteria

and growth

con&ions

Escherichia coli, 1000, F-, su+, thiamine-, was grown in M9 inorganic salts (Anderson, 1946), plus 0.2% glycerol. Cultures were grown at 35°C with shaking in a gyrotary water bath. The mass doubling time was 75 min and cultures were induced at 5 x 10s bacteria/ml. (b) Induction

and labeling

poc-durea

Stable RNA was labeled by adding 0.1 PCi of 2-[14C]uracil/ml (Schwarz-Mann) to cultures at least 2 gcncrations before induct.ion. [5-3H]uracil (Schwarz-Mann, 16 Ci/mmol) was added to give 2 nmol/ml which provided excess uracil during t.he entire labeling period. The Zac operon was inducd 2 min later by adding IPTG (Sigma) to 5 x 10e4 M:. Rifampitin (Schwa.rz-Mann) was added one min after the IPTG to give 200 pg/ml. Cultures were harvested on ice containing chloramphenicol (100 pg/ml) and sodium azide (10 mu). In these conditions complete transcription of the lac operon takes 2.5 min and induction is reduced to less than 10% after 30 s with rifampicin; thus, 4 min after the addition of IPTG a few of the Eat messages will be just short of completion while the remainder were completed in the preceding I.5 min. This spread in age is unavoidable if sufficient Zuc mRNA is to be made. Tho Zuc mRNA must be labeled uniformly. Label was therefore added 2 min before induction to allow pools to approach a constant specific activity. The specific activity of label entering Za.c mRNA was also monitored directly in a control experiment, without prelaboling. A culture was induced for 5 min, to give a constant rate of transcription from the Zac operon, before adding the label. After a delay of 5 s, the labeling of Zuc mRNA, measurd as hybridizable radioactivity, followed an oxponcntial curve with a rate constant equal to that for decay of lac mRNA (Fig. l), showing that even without prelabeling thero was no major variation in specific activity. i Abbreviations used: Message, mRNA coding for a single, functional peptide (a polycistronic mRNA carries several messages); fiG, /I-galactosidase coded from z gene: P. lactose permease coded by y gene; T-4, thiogalactoside transacetylasc coded from a gene; IPTG, isopropyl-P-Dthiogalactoside.

Zac mRNA

145

DECAY

Time(min1 after adding [3H]uracil La1

(b)

FIG. 1. Labeling of Zao mRNA by exogenous [5-3H]uracil. (a) A lo-ml portion of culture was labeled in stable RNA with [14C]uracil and grown to 5 x lo8 cells/ml: IPTG was added and 5 min later, 8 nxnol (160 pCi) of [3H]uracil. At indioated times, I-ml samples were harvested into tubes containing ice, chloramphenicol, sodium azide and 4 ml of unlabeled, uninduced culture. The RNA was isolated from the mixture as described in Materials and Methods up to the DNase treatment, which was at 37W for 30 mm. The sample (2-ml) was then digested with 30 pg of self-digested pronase (Calbioohem, nuolease-free) for 45 mm at 37°C. After addition of NaCl and sodium acetate buffer, the mixture was extracted once with phenol (2 ml), then precipitated by adding 4 ml of ethanol and storing overnight at -20°C. The RNA was recovered by centrifuging and dissolved in 0.5 mI of a mixture of equal vol. of formamide and 6x SSC. Duplicate 200-d samples were hybridized with l/8 sectors of nitrocellulose filters, one with a filter carrying mixed 480 + &3Odkm DNA, and the other with oalf DNA. The cts/min of “Hlabeled 2u.cmRNA in each l-ml oulture sample was calculated from the cts/min hybridized. The line in (a) is given by the equation y = a (t&,2) (1 - 2- (t -o,1)% where a is the initial rate of Zac RNA synthesis in cts/min/min. (b) A second lo-ml portion of the culture, labeled with [14C]uracil and at 5 x lo* cells/ml, received IPTG and 8 mnol (150 pi) of [3H]uracil. Rifampiom was added one min later. Samples were harvested and RNA hybridized as before. To correct for variable losses during purification the sH-labeled Zac RNA was normalized to a constant amount of l*C in each sample.

(c) Preparation

of

RNA

RNA was isolated by a modification of the method of Summers (1970). Cultures (10 ml) were dispensed onto crushed ice with chloramphenicol to give 100 pg/ml and sodium azide to 10 m.~, then centrifuged, resuspended and held for 5 min at 0°C in 05 ml of 0.1 M-T&! (pH S-O), 20% sucrose, 10 mM-EDTA containing 1 mg lysozyme/ml. The sample was made up to 3 ml containing 20 mm-M& and 1.5% sodium dodecyl sulfate, lysing the cells, then treated with cliethyl pyrocarbonate (Sigma, 50 ~1) for 5 mm at 37°C to inhibit nucleases irreversibly (Fedorcsak & Ehrenberg, 1966). 3 ml of saturated N&l at 0°C was added with stirring. The precipitate was removed by centrifugation and then 05 volume of water and 3 vol. of ethanol (- 20°C) were added to the supernatant. After at least 2 h at -2O”C, the precipitate was collected and resuspended in 2 ml of 10 mMTris (pH 7*4), plus 10 mM-MgSO, and treated with 20 pg DNase/ml (electrophoretically purified, Worthington Bioohemicals) for 30 min at 0°C. 40 ~1 each of 1 M-SOdkIm acetate buffer (pH 5) and 5 M-NaCl, then 4 ml of ethanol were added. After at least 2 h at - 20°C, the precipitate was centrifuged out, dissolved in 0.5 ml formamide (Fisher) and incubated at 37°C for 5 min (see below). After adding 05 ml 2 x SSC (SSC is 0.15 M-NE&I, 0.015 m-sodium citrate) the RNA was re-precipitated with 2 ml of ethanol at -20°C.

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If the fo rmamide treatment was omitted, about 5% of the 3H-labeled EcccmRNA sedimented further than expected for full-length molecules. Presumably, this had aggregated; the formamide treatment was included in all experiments reported here. (d) Centrificgation and detection of lac mRNA The RNA was dissolved in O-1 M-N&Y, 10 mu-sodium acetate (pH 5-O), 1 m&r-EDTA containing O*1o/osodium dodecyl sulfate, then warmed to 37°C for 5 min and layered onto a 5% to 20% sucrose gradient in the same buffer without sodium dodecyl sulfate. Centrifugation was in a Spinco SWBOL rotor at about 4°C and 50,000 revs/min for 2.5 h. Samples (170-d) were collected directly into 1 dram vials with rubber lined caps (Wheaton) for RNA-DNA hybridization. A 10-d portion from each fraction was removed for radioactive counting. 70 d of 20 x SSC (to give 230 ~1 of 6 x SSC), 230 d of formamide and a l/S sector of a 25 mm nitrocellulose filter (Schleioher & Schuell, Keens, N.H.) carrying l/8 x 50 pg of denatured, mixed 430 and $80dlac DNA were added. The vials were incubated for 4 days at 45°C; the reaction is then complete. The filters were washed briefly in 2 x SSC, treated with 20 pg pancreatic RNase/ml (Sigma) in 2 x SSC for 60 min, rinsed in 2 x SSC and dried. Duplicate samples reacted with calf DNA alters gave less than 10 cts/min above background (2 to 4 cts/min). Controls showed that sucrose did not affect the yield of hybrid. (e) Other procedures Aqueous samples were made up to 1 ml and counted after adding 3 ml of the scintillation mix of Patterson & Greene (1965). Measurement of radioactivity in hybrid, preparation of the mixed $80 and #SOdlac phage and its DNA for hybridization have been described (Kennell, 1970).

3. Models An RNA molecule might be broken down in any of the following ways: a progressive exonucleolytic attack from the 5’ to 3’ end or from the 3’ to 5’ end; a primary endonucleolytic break at a few, defined targets followed by degradation of the liberated pieces or, fmally, primary endonucleolytic attacks at any of a large number of targets. The secondary degradation of the fragments in the latter cases might be either exo- or endonucleolytic. Since some messages in any population start to decay before they are oompleted (Morikawa & Imamoto, 1969; Morse et al., 19696; Schwartz et al., 1970), exclusive 3’ exonucleolytic attack need not be considered further. There are many possible variations of the remaining three mechanisms but we have considered only three specific situations. The Appendix gives details of the calculation of profiles predicted by the models. In these models we have assumed that decay occurs at an exponential rate from the start of synthesis and that the scavenger degradation of the operator distal fragment occurs at a rate equal to the rate of transcription (nucleotides released or polymerized per min). Both assumptions are supported by earlier observations that in a burst experiment lac mRNA commences to be lost at an exponential rate very close to the time that all the molecules are completed; for the rate to decrease exponentially at this time must mean that molecules are being lost as soon as completed (Schwartz et al., 1970). A more direct measurement can be made from the data of Figure 1. The total counts incorporated in the four-minute period is the net sum of those incorporated at each instant minus their subsequent loss by exponential decay until four minutes. The period was divided into 0*5-minute increments and this summation carried out using the l-5-minute half-life observed, e.g. the l-5 to 2.0-minute period would have decayed by 2.82 half-lives by four minutes. The estimated net

Zac mRKA

DECAY

147

counts in lac RNA by four minutes agreed within 5 to 10% of the observed value ; t,his could only be true if decay commenced with a 1.5-minute half-life at the start of synthesis. It has been shown that RNA polymerases transcribe the lac operon at the same rate, i.e. each requires 2.5 minutes to complete the mRNA (Jacquet & Kepes, 1971). Since the culture had been induced for five minutes before [3H]uracil addition, all parts of the Zacmolecules became labeled simultaneously. The immediate exponential decay at the final steady-state rate shows that all parts of the molecules, including the newly synthesized segments, are vulnerable to decay. (a) Case I. 5’ exonudeolytic breakdown The simplest mechanism involves a progressive exonucleolytic degradation. According to this model (Mosteller et al., 1970), degradation occurs by a progressive exonucleolytic loss of mass from the 5’ end of the mRNA. Such attack might be coupled to the movement of ribosomes (Kuwano et al., 1970). A modification (Forchhammer et al., 1972) suggests that the degradation of individual molecules may occasionally halt. This seeks to explain the slower decay of operator-distal relative to operator-proximal messages observed with trp operon mRNA (Bhmdell et al., 1972; For&hammer et al., 1972). However, chemical loss of the mRNA transcribed from the distal part (about one-third) of the luc operon is markedly faster than t.hat of total lac mRNA at 37°C. The half-lives are 35 and 70 seconds (Pastushok & Kennell, 1974). The faster decay commences as soon as label enters that portion of the molecule and the rate remains constant for at least five half-lives, to as low a level as can be measured with our procedures. For this model we assume: (1) the initiation of breakdown is random; (2) this random attack commences immediately; (3) the degradation rate is the same for all molecules and all parts of a molecule and (4) equals the rate of transcription, 30 to 40 nucleotides/second/molecule. The distribution of radioactive lac mRNA predicted by this model is shown in Figure 2 (curve A). Strikingly, although only 30% of the molecules present remain intact, they account for nearly 60% of the mass (radioactivity), giving the prominent peak. A feature of this model is that degradation causes no increase in the number of mRNA molecules in the cell. Such an increase can only result from endonucleolytic cleavage. A second important consequence of t.he model is that once transcript,ion has ceased, degradation does not change the relative size distribution of the fragments; the mass in each size class is reduced proportionately. (b) Case II. Primary endonuckolytic attacks at the 5’ end of each .me.ssagejolbwed by exonucleolytic degradation A variety of models can be constructed in this case, according to the assumptions made. Two variations are presented; in both, the inactivation of each of the three messages results from attack at the operator-proximal end of the message. In the first variation, every attack is an endonucleolytic cleavage, which exposes the message to a 5’ exonucleolytic degradation similar to that in case I, but which terminates before the start of the next distal message (Fig. 2, curve B). In the second, exonucleolytic degradation, once started, continues to the 3’ end of the molecule, i.e. to the end of the TA message (Fig. 2, curve C). In either case, the frequency of attack on each message decreases at an exponential rate with a unique rate constant. 11

148

M. BLUNDELL

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KENNELL

The known decay rates of the /IG and TA messages in this strain (Kenneh & Bicknell, 1973) were used in the calculations, while the permease (y) message has been assigned an intermediate lifetime. Neither profile shows a peak of intact molecules. Curve B has two peaks, one made up of fragments containing the z gene message and the other fragments remaining after degradation stops at the boundary between oistrons. Curve C has a broad peak containing some intact molecules and a larger number of fragments. Moleculor weight K 10v6

3.0 2.4 1.8 I.4 I,0 0.7 0.4 0.2 0.1 II IIII, II

Fraction no

FIG. 2. Sucrose gradient

profiles of lao mRNA predicted from various models of degradation. It is assumed that bacteria are induced at zero time, rifampicin added at one min and the RNA purilied from the cells at 4 min. Curve A, case I, 5’ exonucleolytic decay; curve B, case II, primary endonucleolytic cleavage at the start of each of the 3 messages followed by 5’ exonucleolytic decay to the start of the next operator-distal message; curve C, case II, s&me as curve B above except that endonucleolytic cleavage at the start of a message is followed by 5’ exonucleolytic decay to the end of the TA message. For details see text and for calculations see Appendix. Approximate molecular weight scale is shown.

(c) Case III. Primmy endonucleolytic attack8 at a large number of internal sites The inactivating event would be an attack by an endonuolease within the message. This model assumes a large number of vulnerable sites in each message. The size distributions from such a model can be derived after choice of various specific assumptions. We have not carried a derivation to completion because there are at least

Zac mRNA

DECAY

149

three reasons why we feel this mechanism for decay is unlikely. First, if one assumes that the targets on a message have about equal sensitivities, then the initial decay of the mRNA would not be exponential. The rate of loss would accelerate at first. In exponential decay the increase in rate of decay is decelerating even though the rate itself may be increasing until foci of degradation are lost. However, as shown above, exponential decay appears to commence at zero time. One can avoid this difficulty by assigning higher vulnerability constants to the targets very near the 5” end. However, the model would then degenerate into the 5’ exonuclease case (model I). An important subset of this model would be one in which all phosphodiester bonds have about the same vulnerability for attack. We have argued against this specific case after observing that there seems to be no correlation between message size and rate of decay; this model would predict that longer messages decay faster because they contain more targets (Blundell et al., 1972). A third argument concerns functional ef6ciency. Each endonucleolytic cleavage would produce two parts in the message: a proximal and a distal. Presumably, ribosomes on the distal part would continue to translate and produce functional polypeptides while those on the proximal fragment could either continue translation to the end of the fragment or be stranded in place leaving the cell with the problem of disposal of the complex. The amount of incomplete peptides produced in the first instance would be twice those produced if they were stranded. This follows since at the time of cleavage the peptides range in size from one to n amino acids (0.5 n average) while if they all were to grow subsequently they would reach n size. The minimal fractional mass of incomplete proteins that would be produced can be estimated (see Appendix). If ribosomes were to complete translation of the proximal fragments, then more than 25% of the polypeptide mass synthesized by the cells would be incomplete. If ribosomes were stranded, this figure would be greater than 10%. A much smaller fraction of E. co& protein is unstable in growing cultures (2 to 7%, Nath & Koch, 1970; 26 to 3%; Pine, 1970). These could represent incomplete molecules but would only account for a part of those produced in this model; the remainder would be present as non-functional polypeptides in the cell. 4. Results A specific mRNA would best be characterized by purifying it and then measuring its size and other characteristics. However, attempts to isolate lac mRNA by hybridizing to DNA and subsequently melting the hybrid failed because, even at low temperature (35°C) in the presence of formamide, there was an unacceptable level of hydrolysis. Thus, we had to reverse the procedure and fractionate total RNA by sucrose gradient centrifugation before measuring the amount of lac mRNA in each fraction by hybridization. The sucrose gradient profile of lac mRNA radioactivity from cells harvested four minutes after adding IPTG is shown in Figure 3. This profile may be compared with the predicted profiles in Figure 2. The observed patterns agree most closely with Figure 2, curve C, although as opposed to the model, they show somewhat more full-length than two-thirds length lac mRNA. Inaccuracy in any number of minor assumptions could account for the difference. For example, since the RNA shifts to smaller size upon chase, the comparative enrichment for full-length molecules could result from

160

M. BLUNDELL

AND

Molecular

D. KENNELL

weight x 10e6

I.6 I.5 I.2 0.9 0.6 III,,

03 I

IO

0.1 I

20 Fraction

no.

Fro. 3. Sucrose gradient centrifugation of Zac m.RN-4 from a sample harvested 4 min after induction. [sH]urilca was added 2 min before and rifampicin one min after inducer. --O--O--, Standard procedure (see Materiels and Methods); -O--O-, cells left an additiona 2 h before lysis; standard procedure except for omission of IPTC; is indicated. 14C in stable RNA is shown; the W proflos in the 3 gradients were similar and that from gradient (--a--@--) is shown.

the finite time for complete block of initiation by rifampicin. However, the observed pattern shows no similarity with that from case I; this is shown in Figure 4 where the observed values are normalized to give 100% for the entire gradient. There is no pronounced peak corresponding to full-Iength molecules (1.75 million daltons) as predicted by the 5’ exonucleolytic model (case I) ; much more radioactivity is present in regions of the gradient to which smaller molecules sediment. This result appears to be inconsistent with an exclusive 5’ exonucleolytic degradation of lac mRNA. Conceivably, the experimental profile could be generated by endonucleolytio activity or shear during the isolation of the RNA. Three lines of evidence argue against this possibility. First, controls show that ltzc mRXA is not degraded during isolation. Two phases of the isolation procedure were tested: the period when the RNA is still in the cell, and the period after lysis. Between harvesting and lysis, RNA remains in the cells for about 40 minutes at 0°C in the presence of chloramphenicol and azide. To show that no significant cleavage occurs during this time, a parallel sample of culture was loft for an additional two hours before centrifugation and lysis. Sometimes

Zao mRNA

151

DECAY

Molecular weight x IO-’

3.0 2.4 IIS 14 I.0 O-7 O-4 02 01 r" " " ' "

I I

0 I

I

t i 5’- Exo 1 I : : i : : \.

:

: 23

16

b Fraction no.

Pm. 4. Comparison of an observed 4 min prof& with that predicted in case I (5’ exonuoleolytic). Profiles of Fig. 2, curve A, and RNA from cells harvested 4 min after induction as in Fig. 3 plotted on the same scale. The total predicted or observed cts/min are normalized to lOOo/ofor comparison. The molecular weight assignments calculated in the Appendix for the gradients of Fig. 3 are also shown.

there was a slight, unexplained increase in the radioactivity in high molecular weight luc mRNA (as shown in Fig. 3) but there is no evidence of cleavage. To show that no degradation occurs during lysis and the subsequent purification, ZacmRNA was subjected to a second cycle of isolation. Gradient fractions containing high molecular weight [3H]RNA were pooled. A second culture was labeled in stable RNA with [14C]uracil, but received no 3H before the usual induction and lysis. One half of this lysate was mixed with about two-thirds of the pool of [3H]RNA. The RNA was purified from both lysate samples as usual and then the remainder of the [3H]RNA pool was added to the RNA from the other half of the lysate. Thus, the first part of the [3H]RNA was exposed to the purification procedure for a second time, while the second part was not and served as control. After a final ethanol precipitation, the two samples of RNA were centrifuged through gradients and fractions hybridized. The two profiles were similar (Fig. 5) and the “H-labeled lac mRNA sedimented at the same rate as before. Thus, the lac mRNA survives undamaged through the isolation procedure. In this particular experiment the DNase treatment

was omitted

to test

for possible

contaminating

endo-RNase

activity

which had been reported by Bramwell(l972) to be present in this commercial DNase. A third sample was treated with DNase and gave an identical pattern; there appears to be no RNA degradation from the DNase treatment. Second, even if there were significant, cleavage during purification of the RNA,

162

M. BLUNDELL

AND Molecular

D. KENNELL

weight x 10e6

22 18 I.4 IO 06 I I 1--v---

02 01

Fraction no.

FIG. 5. Control for degradation of kzc mRSA during purifioetion. -@-a--, High moleculer weight Zac mRNA from sucrose gradient fractions indicated by the indent shown wa.s mixed conk01 portion from the same Zuc mRNA pool with fresh lysete and repurified; --c--C--, added to RNA from fresh lysete just before centrifugation. (-------) A portion of the repurified RNA was treated with DSase (10 pgglml) at 37°C for 30 mm beforc centrifugation. Results from 3 gr8dkntS centrifuged at the same t.ime are shown. The gradients contained 14C-labeled stable RNA; the ‘*C pro6les were similar and the profile from the re-cycled RSA (no DBase) is shown. The “spread” of RNA to adjacent fractions is due both to the presence of smaller moleoules in the origin81 pool and to the “spread” of all molecules to adjacent fmctions after centrifugation. An approximate molecular weight scale is shown.

it is unlikely t,hat the observed profile would be generated from the mRNA population predicted in case I. Neither shear nor random endonucleolytic attack would be expected to produce the large number of small molecules found while still leaving significant quantities of much larger molecules. Third, model I (exclusive 5’ exonucleolytio breakdown) predicts that the profile of radioactivity will remain unchanged, except in scale, at later times (see Discussion). In contrast, endonucleolytic attacks will generate a population of smaller molecules with time. Figure 6 shows the profiles of samples harvested at la,ter times. For comparison of relat.ive size distributions, the [3H]RNA4 hybridized in each gradient is plotted on a percentage scale. Note that the total lac RNA is being lost exponentially, so there is much less radioactivity in the later samplos. As the

Zac mRNA

153

DECAY

Molecular weight x 10s6 I.8 1.5 I.2 0,9 0.6 IIII I

0.3 I

0.1 I

IO -

8gi 2 ’ 68 * 22 i% 9 f 4-

Fraction no. (a)

(b)

FIG. 6. Comparison of sucrose gradient profiles of Zuc mRNA from samples harvested at various times after induction. [3H]uracil was added 2 min before and rifampicin one min after inducer. 14C in stable RNA was present in each gradient to identify the 16 and 23 S positions. (a) Samples from the same flask harvested at 4, 6, 6, 8 rmd 10 min after induction. The stable RNA markers sedimented to the same position in each gradient, shown by the arrows; 4 min (--e--O--); 5 min (-O--O-); 6 min (--m--m--); 8 min (-A-A-) and 10 min (--A--A---). Total 3H-labeled Zac RNA hybridized in each gradient was at 4 min, 38,000 cts/min; 5 min, 21,500 cts/min; 6 min, 11,000 cts/min; 8 min, 4000 cts/min; 10 min, 2000 cts/min. (b) Shows 4 min (--e--O--) and 6 min (-O-O-) patterns from a different experiment in olearer contrast.

Zac mRNA decays, there is a marked shift to smaller molecules. In particular, we a persistent peak containing fragments about two-thirds of the length of the largest molecules. These could be x messages liberated by cleavage between the x and y cistrons. Figure 3 shows another control, the profile of a sample of culture treated by the standard procedure except that no IPTG was added. Con&se et al. (1970) reported that uninduced E. coli contains a substantial amount of luc mRNA, almost entirely of quite small size, We could find no evidence for enrichment of smaller molecules. The difference in results may be due to significant non-specific binding iu their experiments. Using our conditions, reaction to heterologous DNA gives essentially background counts bound (see Materials and Methods).

notice

154

M. BLUNDELL

AND

D. KENNELL

5. Discussion (a) Are incomplete lac messeager RNA molecules degradation prochcts? The experimental procedure applied here allows time for all polymerases to complete transcription of the lac operon (Jacquet & Kepes, 1971). This procedure was adopted so that all incomplete lac mRNA molecules would be degradation products. Recently, DeCrombrugghe et al. (1973) have shown that mRNA transcribed from the lac operon in vitro is terminated prematurely in the presence of a high concentration of p protein (Roberts, 1969). They suggest that natural polarity might be explained by transcriptional termination within the operon. However, the observed termination occurs about one-third of the way along the .z gene. It seems unlikely that such partial transcriptions can produce any active enzyme. It has been known for some time that certain pairs of point or deletion mutants in z can complement in vivo or in vitro to give PG by a non-covalent association of polypeptide fragments. However, the resultant enzyme is markedly different in physico-chemical properties from normal j3G and only about 1% of the enzyme from wild-type E. coli contains these free a or w complementing subunits (Ullmann & Perrin, 1970). Furthermore, a variety of chemical studies have suggested that the monomer for j3G is a single polypeptide of 135,000 molecular weight (Zipser, 1963; Karlsson et al., 1964; Brown et al., 1966), as have co-linearity correlations between various nonsense mutants (Newton, 1966) and the size of their polypeptide products (Fowler & Zabin, 1966) ; (see review by Zabin & Fowler, 1970). The evidence seems very strong for a single 2 cistron. To account for the approximately threefold (Zabin & Fowler, 1970) natural polarity, two out of three transcriptions would have to be terminated between the z and a genes. Our results do not support this explanation, since we observe significantly more full-length than two-thirds length lac molecules at four minutes. Furthermore, at 37°C the a message decays two to three times faster than the x message (Kepes, 1967; Kennell & Simmons, 1972) and this, in itself, can account for the decreased yield of TA compared to /3G (Blundell et al., 1972). Another potential source of incomplete lac mRNA molecules should be considered. If some RNA polymerase molecules transcribed more slowly than others, then some incomplete transcripts would be observed at four minutes. However, Jacquet & Kepes (1971) showed, by measuring the effect of actinomycin D on ,&galaotosidase synthesis, that all polymerases transcribe at least the x gene of the lac operon in the same time. Finally, it should be noted that the shift to a smaller size distribution when the luc messages are chased (Fig. 6) obviously results from degradation and would be inconsistent with the 5’ exonucleolytic model even if there were incompIete transcripts present at four minutes ; such molecules would also show an unchanged distribution of sizes during their decay. The possibility that decay might slow significantly or stop has been considered and eliminated above (see Models). (b) Evidence against exclusive 5’ exowwleolytic decay Earlier observations suggested that the temporal sequence of breakdown of the trp mRNA (Morikawa & Imamoto, 1969; Morse et al., 1969a) couId simply be a necessary consequence of the temporal sequence of synthesis from the 5’ to 3’ end.

Zac mRNA DECAY

155

The functional inactivation rates of different messages differ significantly even for messages in the same polycistronic molecule (Blundell et d., 1972). This suggests that each message is attacked independently. Cases in which a distal message decays faster than a proximal one, as for the lac operon at 37°C (Kepes, 1967; Kennel1 & Simmons, 1972; Bhmdell et d., 1972) can only be reconciled with exclusive 5’ exonucleolytic degradation by invoking a second, non-degradative mechanism to inactivate the operator distal genes. Even the temperature coefficients of inactivation within the kc operon can differ markedly, again suggesting independent targets (Kennell & Bicknell, 1973). While the foregoing observations are suggestive, the data in this paper show directly that the lac message cannot be destroyed by sequential breakdown only. The size patterns shown in Figure 3 are very different from those predicted from the model of exclusive 5’ exonucleolytio decay shown in Figure 2. Even if it is assumed that the controls are inadequate and the patterns reflect hydrolysis during preparation, there is an independent observation that is also inconsistent with this model. If degradation was by 5’ exonucleolytic attack alone the distribution of lac mRNA among different size classes would remain constant after transcription had ceased (at 3.5 min). As the population was degraded, the quantity of molecules in each size class would be reduced in proportion, by a factor of 2t’t* (see Appendix). The patterns observed at later times (Fig. 6) clearly differ from the four-minute pattern. Unless it is argued that, despite the controls, the RNA is damaged during extraction and also that RNA extracted after later times consistently suffers more than does RNA extracted at four minutes, this result can only be explained by endonucleolytic attack during messenger decay. The results presented above cannot distinguish either the number of sites sensitive to endonucleolytio attack, their relative sensitivities or their distribution. Cleavages within a message would lead to production of incomplete peptides which could become a significant fraction of total peptides synthesized (see Models). If the targets are restricted to the start of each oistron, this problem is avoided. Inactivation of a ribosome loading site or an untranslated region between messages would prevent further initiation of translation but allow ribosomes already on the message to make complete proteins. Such a mechanism brings to mind the mechanism proposed for polarity (Morse & Yanofsky, 1969). However, polarity can be partially relieved by certain suppressor mutations such as SUA(Beckwith, 1963; Scaife & Beckwith, X966; Morse & Primakoff, 1970) and others (Carter & Newton, 1971), but suA has no obvious effect on the decay rate of trp mRNA (Morse & Primakoff, 1970), lac or total mRNA (Kennell & Simmons, 1972) during normal growth. The factors which control the sensitivity of the various sites are so far undefined although deletions of the lac operon (Blundell et al., 1.972) and the trp operon (Forchhammer et d., 1972) can alter the decay rates of messages from the distal genes. The rate of attack at each site could depend on the secondary and tertiary structure of the mRNA molecule. Thus, while the evidence for primary endonucleolytic attack appears strong, the detailed mechanism of messenger RNA decay remains obscure.

156

M. BLUNDELL

AND

D. KENNELL

APPENDIX Phy.sicul parameters

The size of lac mRNA and its constituent messages has been estimated from the sizes of the peptides for which it codes. The x gene contains 3500, the y and a genes about 900 base pairs each (calculated from Zabin & Fowler, 1970; Jones & Kennedy, 1969). The total of 5300 will be transcribed into an mRNA of 175 million daltons. In this strain at 3i’“C, the chemical half-life of Zac mRNA is 15 minutes (Fig. 1) and the functional half-lives of the /3-galactosidase and transacetylase messages are 15 and 075 minutes, respectively. The decay rate of the permease (P) message is not known and a half-life of one minute has been assumed. Transcription of the PC: gene takes 1.5 minutes and of the entire operon 2.5 minutes. The P message should therefore be complete at about 2.0 minutes. Calculation

of molec&ar weights in sucro’osegradients

The relationship between szo.W and molecular weight for single-stranded RNA at a given temperature and ionic strength should be that typical of a random coil (Boedtker, 1968; Fresco & Doty, 1957): s20*w = #mlO’45.

(1)

In the rotor and gradient used, the distance sedimented is proportional to s20,Wand time (Martin & Ames, 1961). The average molecular weight in each fraction of a gradient was therefore calculated from the positions of the peaks of 23 S, 16 S and 4 S [14C]RNA (1.1 x 106, 5.5 x IO5 and 2.5 x 104, respectively) in the gradient, using the equation : iM = (a - bF)2.22, (2) where F is the fraction number. Since the two major markers, 23 S and 16 S RNA, lie in the center of the range of interest, a moderate error in equation (2) would have no significant effect on the assignment of molecular weights. However, one sonrce of uncertainty which cannot be evaluated is the effect of secondary structure on sedimentation. Ribosomal RNA has considerable secondary structure, while that of lac mRNA is unknown. Nevertheless, the fastest moving lac mRNA, presumably intact molecules, is found where predicted using the rRNA species for reference. After sedimentation and collection, RNA species which are homogeneous, such as the 23 S and 16 S rRNA, are not confined to single fractions. This spreading must be largely the result of mixing since such large molecules diffuse very slowly (the diffusion coefficient of 23 S RNA is lo- ’ cm2/s). To correct for this effect and thereby produce realistic gradient profiles representing the theoretical size distributions, the theoretical radioactivities were redistributed according to the shape of the rRNA peaks. Thus, one unit in fraction (n) was distributed to give O-085 unit in both (n - 2) and (n + 2), 0.24 unit in (n - 1) and (n + l), leaving 0.35 unit in fraction (n). Case I. Exclusive 5’ exonucleolytic degradation To account for the exponential kinetics of both inactivation and chemical loss, the initial attacks at the 5’ terminus of the mRNA must be random and followed

tat

mRNA

157

DECAY

by oontinuous degradation destroying the molecule at approximately the same rate as transcription (see Models). The degradation rate, D, is therefore assumed to be equal to the transcription rate, 5300/2-5=2100 nuoleotides/min, and also constant along the molecule. The rate at which molecules are attacked to initiate degradation, A, is given by: dA - = kN, dt

(3)

where N is the number of unattacked 5’ termini present at time t and: k = ln,2/t+ = 0.462 min-I.

(4)

With inducer added at zero time and rifampicin at one minute, the mRNA term% in the culture increases during the first l-5 minutes, cules attacked in this initial period will be completely degraded within Since no samples were taken before then, the intact 5’ termini present may be taken as the base for calculation (IV,). From this time (t = dA’ -= dt

dA --=dt

kN

number of Eat but any molefour minutes. at 1.5 minutes 1*5), (5)

and therefore : N

=

Noe-k't-1'5'

(6)

From the moment of attack, the length of an individual molecule decreases by D nucleotides/minute. A molecule attacked at time t, will be shortened after an additional time, t,, to length: L = 5300 - t,D. (7) The fraction, G, of molecules longer than L (0 < L < 5300) at time t, + t, is the fraction unattaoked at t,, equal to Q = N/N, = e-k(tl-l+)o

(8)

At time t, the value oft, for any chosen length is given by t, = t - t, = t - (5300 - L)/D ...

G

=

(9)

e-k(t+(L-5300)/D-1.5)

(10)

But 5300/D = 2.5 ...

G

=

,-k(t-4+L/D,

=

2-(t-4+LIDNt).

(11)

Equation (11) may also be written : ,‘J =

e-kLID

x

e-k(t-4)

Thus, the relative distribution of sizes fraction, G, for molecules greater than (emkct- 4)) as do all other fractions. The obtained by putting L = 5300. For the gradients of Figure 3, equation

=

2-LI’Dxtf)

X

2-U-4,&.

(12)

remains unchanged with time since each some size L, changes by the same factor fraction remaining intact at any time is (2) becomes :

M = (890 - 30ly.22.

(13)

:58

RI. BLUNDELL

ASD

D. KENNELL

The average molecular weights at the center and boundaries of each gradient fraction are calculated from equation (13), then the boundary values are divided by 330 to obtain L in nucleotides and subst.ituted in equation (11). The dXerence between t.he two values of G gives the number (X/nT,,) of molecules in the fraction. This is now multiplied by the average molecular weight in that fraction to give the mass of message in arbitrary units. Finally, the mass in each fraction is redistributed as described above, producing the profile in Figure 2, curve A. Case II. Primary endonucleolytic clemage at three sites According to this model (Blundell et al., 1972) there is a region at the start of each message, including the first (BG), w h’ic h is the target of an entlonucleolytic attack that is followed by exonucleolytic degradation. The rate of endonucleolytic attack would depend on the structure of the mRNA, so that each message would have a unique decay rate. If the progressive 5’ exonucleolytic degradation is unable to continue from one message to the next, the rate of cleavage at the start of the P and of the TA message must equal their respective inactivation rates. Consider a population of molecules initiated at time zero. At four minutes, the site of /3G will have been in jeopardy for the whole four minutes, the P site, synthesized 1.5 minutes later, for 2.5 minutes and the TA site for 4 - 2 = 2 minutes. Therefore, the fraction of each message which remains unattacked and active : A = N/No

= e-k’

=

2-t/b,

(14)

mill be l5*8o/o for /?G, 17.774 for P and 15*8o/o for TA. Similarly, for a populat.ion initiated at one minute (at the time rifampicin was added) the figures are 25*00/,, 354:/o and 39.70/,. Using these fact.ors, t.he fraction of molecules with hits at each of the eight possible combinations of sites (none, /3G or P or TR only, PG & P, /3G & TA, P & TA, all three) can be calculated. Each of the fragments produced by the endonucleolytic at,ta.ck is degraded by the mechanism of case I and is therefore treated by using equation (1 l), with a refinement for species consisting of more than one message since they will be degraded only to the start of the next message. Thus, the size of the distal part that remains defines the minimum size (B) at which degradation stops. Equation (11) then becomes: G

=

N/1\;,

-

e-k(t-t'+WB)iD),

(15)

whore t’ is the time taken t;o transcribe to the 3’ end of t,he message being degraded, and is obtained in the same way as the 2.5 minutes substituted in equation (10). To allow for the range of initiation times, ten separate calculations wore carried out, for populations initiated at six-second intervals. The number of molecules falling in each fraction were added together and treated as in case I, to give the profile of Figure 2(b). .If the progressive 5’ cxonucleolytic degradation can continue into operator+listal messages, their inactivation will be the sum of exo- and endonucleolytic att,acks. Consider the operator-proximal end of the P cistron in a populat,ion of molecules initiated at time zero. Since t.he P message has been inactivated exponentially from the moment its 5’ end was transcribed (I.5 min after the start of the /3G message), the fraction remaining active is :

Zac mRNA A,

_

DECAY

159

e-kD(t-l.5),

(16)

where the time of luc mRNA decay, t, is 21.5 and k, = 0.693. Now, some of t.hese P messages were inactivated exonucleolytically. This happened when the adjacent fl message was degraded completely, and since it requires 1.5 minutes for the /?G decay to reach P, the fraction of /3G messages which have not yet been degraded completely is given by: ,I&

=

e-k,m(t-l~5),

(17)

where again, t 2 l-5 and kga = O-462. The fraction of active P sites (A,) plus the fractions inactivated by ,9G hits (B,) or by endonucleolytic attacks at P (F,) must add up to one: Ap + B, + F, = 1, and since 1 B, = EBG, F, = EBo -

A,,.

(18)

Similarly, the fraction of molecules with enclonucleolytic attacks at the TA site, which is first made at two minutes after the start of the Inc mRNA, PTA

=

E,

_

A,,

=

e-kp(t-2.0)

_

e-kTA(f--2.0)e

(19)

Endonucleolytic cleavage at the P site generates fragments ending at the 3’ end of the z gene. Some of these will be undergoing exonucleolytic degradation, and these z gone fragments: can be treated by applying equation (11) for 0 < L < 3500. The fract,ion of t.hese fragments in which the z gene remains intact is given by L = 3500. The other fragment generated by the cleavage, carrying the P and TA messages, will be degraded exonucleolytically. It joins, and is indistinguishable from, the population of molecules generated by oxonucleolytic attack originating from the /3G target. Once the P message has been inactivated, nothing differentiates the P - TA fragments generated by exo- and endonucleolytic attack. Thus, the rate of exonucleolytic attack on the TL4 messa.geis exponential w&h t.he rate constant of P message inactivdion (0.693). Enclonucleolytic attack at the TA sit.0 liberates fragments containing alllor part of the 1’ message and sometimes all or part of the /?G message. What is their size distribution? Beca,use endo attacks at the P site generate the class of molecules already considered, the size clist.ribution of fragments cut at t,he TA site will be given by applying equation (11) for 0 < L < 4400, using the rate cordant of P message inactivation (0.693). In other words the fraction of molecules of size >L will be decreasing both by cndo hits at P that simply eliminats the fragments from t,his class a.nd by the exonucleolytic decay from the /3G site which shortens them. The sum of these Owo decay processes gives the P inactivations. Similarly, the size distribution of fragments carrying t,he TA message can be obtained using 0 < L < 5300 and the rat,e constant of TA inactivation. Case III. Production of incomplete peptides by endonucleol~ytic cleavage at nzany site8

Cleavage at an interna. site will yield a proximal and distal part of the message. In the following calculation we =sume uniform vulnerability of many equallyspaced ta.rgets and equa,l spacing of ribosomes along t.he message. After a cleavage, ribosomes cannot cont,inuc loading onto the operator-distal portion. However, the original loading site is still present on the proximal portion and ribosomes would

160

M. BLUNDELL

AND

D.

KENNELL

continue to reassociate if translation of this fragment were to continue. It might or might not continue, so both cases have been considered. We have estimated fractional mass of incomplete peptides for any half-life period assuming that both proxime1 and distal parts are lost in that one half-life. In any given half-life period (15 min) subsequent, to completion of the z message of L nucleotides, there are three sources of peptides. (1) Half the message will have received hits and assuming continued translation, the operator-proximal portion will make as many peptides in the l-5minute period as would a full-length molecule since ribosomes can continue to load at the start; this is defined as proportional to (L/L). The mass of each peptide would be proportional to l/L, where 1 is the length of the proximal fragments. (2) The number of complete pcptides from the operator-distal portion would be proportional to (L - 1)/L since rihosomes cannot continue loading onto the distal part. They would be completed with mass proportional to L/L. (3) Half the messages will have received no hit. They will produce a number and mass of peptides each proportional to L/L. Thus, the fractional mass (F) of peptides that are incomplete is:

where k is a constant. :. F = O-25. If ribosomes were stranded on t.he proximal fragments, the number of incomplete peptides would be proportional to l/L with average mass proportional to 4 l/L to give : F = 0.10. During synthesis, half of the z messages are hit (t+ = 15 min). The amount of incomplete peptides synthesized would be the same as in subsequent half-lives. However, the number of complete peptides made from the dist.al portion would be a complex function of 1 and the time of attack since ribosomes cannot reload on this part. Limits would be between zero (time of attack equal ta time of target synthesis) and L/L (time of attack at 1.5 min) and can be used to estima,te limits of F. The amount of complete peptides from unhit molecules would be the same as in subsequent half-lives. With continued translation of the proximal part: O-25

< F < 0.33.

If ribosomes were stranded on the proximal pieces : 0.10 < F < O-14. !&us, t.hc total incomplete polypeptides would account for 10 to 33% of total protein synthesis. F will vary only marginally its a function of message size and half-life since its value is so similar in the synthesis and post-completion phases of message translation. Thus, these values would apply to total cell protein. This investigation was supported by rusearch grants GB19198 from the National Science Foundation as well as GM19375 and Research Career Development Award (I-KO-4-GM06688) to one of us (D. K.) from the National Institutes of Health. Daniel Achord and Louis Lim participated in several experiments. Computation was done on the Cambridge University Titan Computer.

Zac mRNA

DECAY

161

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