J. Mol. Sol. (1975) 92, 311-318

Differential

Modes of Chemical Decay for Early and Late Lambda Messenger RNA

RADHEY SRYAM GUPTA AND DAVID

SCHLESSINWR

Departmerd of Microbiology Wmhington University X&o01 of Medicine Xt. Louis, MO. 63110, U.X.A. (Received 22 July 1974, and in, revised form 24 October1974) In the presence of rifampicin, chemical decay of pulse-labeled phage X “early” messenger RNA ocourredexponentially, with a half-life of 2.2 minutes at 42Q compared to 1.3 minutes for the host mRNA. In contrast, mRNA synthesized late during A development exhibited a characteristic complex decay curve not previously observedfor microbial mRNA; initial slow decay of about 25% during 4 to 4.5 minutes was followed by rapid exponential decay of the remainder with a half-life of I.5 minutes. Sucrose density-gradient analyses of the RNA revealed that during the phase of slow decay, the h-specific mRNA was progressively fragmented.

1. Introduction Many phage messengerRNAs are much more stable than the average mRNA of Escherichia COG;for example, T4 (Craig et d., 1972), $X174 (Hayashi & Hayashi, 1968), Ml3 (Jaenisch et al., 1970), and 513 (Puga et al., 1973) mRNA show a chemical half-life two to threefold longer; T’7 (Summers, 1970; Marrs & Yanofsky, 1971) and R17 (Hattman & Hofschneider, 1967) mRNA are even more stable. In all these cases, it has been inferred that the greater stability reflects some intrinsic feature(s) of the structure of mRNA species,since the host mRNA is lessstable in the very same cells. Recently, however, evidence has been accumulating (Forchhammer et al., 1972; Yamamoto & Imamoto, 1975; Silengo et al., 1974) to show that even the same sequence of trp mRNA can show widely different rates of degradation in growing cells. The case of htrp mRNA is best worked out; trp transcripts formed by readthrough from a h promoter are stable (half-life >20 min), whereas the same trp mRNA sequencepromoted by the usual trp starting sequenceare degraded in several minutes (Yamamoto 85Imamoto, 1975). These results suggestedthat the association of trp with h phage-specific sequences leads to stabilization of an mRNA sequence. Two distinct possibilities are: (1) the X mRNA is intrinsically stable and this stability is extended to other sequences attached to it, and (2) some h-specific factor (e.g. products of early genes like N, tof, etc.), has an mRNA stabilizing activity. To begin to distinguish between these alternatives, we report here someanalysis of the decay of bulk h mRNA in vivo at intervals after the induction of phage in a temperature-inducible lysogen.

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and Methods

(a) Bacteria and culture conditions E. coli W3110 (Xc1857 57) (a gift from Dr H. Lozeron) was grown at 30°C with shaking in minimal salts medium supplemented with 1% Difco technical grade Casamino acids, 025% glucose, tryptophan (50 pg/ml) and thiamine (10 pg/ml). (b) Preparation of phage and phage DNA Phage /\ development was induced by heat in exponentially growing cultures of E. coli W3110 (&I857 57). To a culture at 3O”C, an equal volume of warm medium at 60°C was added and the flask transferred successively to a water bath at 42°C (for 15 mm), followed by a bath at 37°C (for 4 h). The bacteria were then lysed with chloroform. Phages were purified from the crude lysate by (NH&SO, precipitation followed by banding to equilibrium in a CsCl step gradient (Kaiser & Hogness, 1960). DNA used in hybridization assays was prepared by (thrice) repeated phenol extraction of the purified phage. The aqueous phase was dialyzed against 1 X SSC (0.15 M-NaCl; 0.15 M-sodium citrate, pH 7.0) for 24 h with at least three changes. (c) Protocol to naemre the rate of RNA decay after tifampicin addition. A 25 ml culture of E. coli W3110 (ho1857 S7) was grown for two generations in the presence of [14C]uraoil (O-1 &X/ml, spec. act. 61 mCi/mmol) to prelabel total cellular RNA. After this pulse-labeling and long chase period, the prophage I\ was induced by adding an equal volume of medium at 60°C and the flask transferred to a 42°C bath. At an appropriate time, as indicated in the text, 1 mCi of [3H]uridine (spec. act. 43 Ci/mmol, Radiochemioal Centre, Amersham) was added to the culture. After 1 min, rifampicin (10 mg/m.l in 50% ethanol), nahdixic acid (1 mg/ml), and cold uridine (5 mg/ml) were added to the culture to give final concentrations of 300, 20 and 200 pg/ml, respectively. The time of addition of rifampicin is considered to be zero time. Thereafter, 3-ml samples were removed at intervals and added to 10 ml of semi-frozen minimal medium containing 20 mrvr-sodium azide and 150 pg chloramphenicol/ml. Cells were washed twice with the above medium by oentrifugation, and were then resuspended in 2 ml of 0.02 M-sodium acetate (pH 5.5); 1 mu-EDTA; 0.5% sodium dodecyl sulfate (Matheson-Coleman & Bell). RNA was extracted twice with phenol, first at 60°C and then at room temperature. The aqueous phase was made 0.1 M in KCl, and the RNA precipitated with 2.5 vol. of ethanol at - 20%. The precipitate thus obtained was dissolved in 0.01 nn-TriseHCl (pH 7.5) containing 0.01 M-MgSO,, and treated successively with 10 pg DNAase/ml (electrophoretically pure, Worthington Biochemicals) for 30 min at 37”C, followed by treatment with 100 rg Pronase/ml for another 30 min. The Pronase was preincubated for 30 min at 37°C at 2 mg/ml before use, to remove any contaminating RNAase activity. RNA samples were once again extracted with phenol, precipitated with 2.5 vol. of cold ethanol, and stored at -20°C. (d) Hybridization. procedures Filters used in hybridization studies were prepared as follows: h DNA was diluted in 1 x SSC to a concentration of 50 pg1m.l and then was denatured by the addition of an equal volume of 1 N-NaOH. After 1 h at room temperature, the flask containing the DNA was transferred to an ice bath and the solution was neutralized to pH 8.5 to 9-O with O-5 N-HCI. An equal volume of 6 x SSC was then added and 10 ml of this solution (i.e., 62.5 pg DNA) was filtered through a membrane filter (Schleioher & Schuell, New Hampshire, 27 mm diameter; 0.45 pm pore size; lot 4252) with low suction. The filters were washed with 40 to 50 ml of 3 x SSC and then air dried. Each filter was cut into 8 equal sectors, which were then incubated at 80°C for 2 h. For the assay of X-specific mRNA, 50 to 250 ~1 of an RNA preparation was brought to O-5 ml with 6 x SSC in a 6 mm x 50 mm test tube. Filter bits bearing X DNA were added, and controls were set up in an identical manner with filter bits bearing no DNA. Each tube was securely covered with Parafllm and incubated for 20 h at 66°C. Filter bits after hybridization were washed with 30 to 60 ml of 6 x SSC and then treated with pancreatic

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RNAase (40 pg/ml) in 2 x SSC for 1 h at 37°C. After several washings with 3 x SSC, the filters were dried and counted. Control experiments in which the RNA to DNA ratio was varied over a lo-fold range showed that the fraction of RNA hybridized remained constant under these conditions, so that DNA was in excess. (e) Sucrose den&y-gradient analysis 10% to 30% linear sucrose density gradients were made in buffer containing O-1 M-T&/acetate (pH 7.5); 5 mM-EDTA, 0.5% sodium dodeoyl sulfate. 0.25 ml of an RNA preparation in the above buffer was layered on the gradient and centrifuged at 4’6 in an SW2’7.1 rotor for 30 h at 25,000 revslrnin. About 30 fractions were collected from each gradient by pumping from the bottom. 100-g portions were counted directly or used 17-ml

in hybridization

assays.

3. Results (a) Decay kinetics of lambdaand host messenger RNA in the presenceof rifam@ci% Soon after infection with X (or induction of a lysogen), some of the phage genes, the “immediate early class”, are transcribed (Skalka, 1966). Products of these immediate early genes then in turn switch on the transcription of the “delayed early” and “late” genes (Skalka, 1966). The shift in transcription from “early” to “late” occurs between three and five minutes after infection at 42°C;

I0

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FIG 1. Decay kinetics of E. coli and h mRNA at 42°C in the presence of rifampicin. Bacteria prelabeled with [W]uracil were heat-induced and then pulse-labeled with [sH]uridiue at 0 mm, 3 mm (h early), or 15 mm (X late) after induction. 1 miu after the addition of [3H]urid.ine (0 time), rifampicin, nalidixic acid and unlabeled uridine were added to the culture to give final conms of 300, 20 and 200 &ml, respectively. Samples were removed at subsequent times and RNA was extracted. The half-life of E. co& mRNA was calculated from the trial in which pulse-label was added at 0 min. It was estimated from the ratio of sH/W at various times. (The ratio in stable RNA observed after all unstable RNA had decayed was subtracted from the 3H/14C ratio at each earlier time to give estimates for the ratio in unstable RNA.) The decay of early and late h mRNA was estimated from the trials in which label was added at 3 and 15 mm, respectively. At each time, samples of radioactive RNA were hybridized to X DNA and the counts hybridized in eaoh case were normalized to a constant input of RNA (by its content of 14C cts/miu). --l --- l ---, E. COG mRNA; -X-X-, h early (3 mm) mRNA; --O--O-, h late (15 mm) mRNA.

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and by 15 minutes, late species comprise the bulk of the h mRNA (Kourilsky et al., 1971; Skalka, 1966). In the experiments reported here, phage development was induced by heat in cells of E. coli W3110 (h&357 X7) (a lysogen with a temperature-sensitive repressor). Figure 1 shows the results of a standard type of trial in which the decay of pulselabeled early and late X mRNA in cells was measured after the addition of rifampicin. Early and late classes of X mRNA were pulse-labeled with [3H]uridine at three and 15 minutes after induction at 42”C, respectively. One minute later, rifampicin (300 pg/ml) and unlabeled uridine (200 pg/ m 1) were added to inhibit further incorporation of [3H]uridine into RNA. h-specific mRNA was detected by DNA-RNA hybridization of RNA extracted at each indicated time. As seen in Figure 1, mRNA pulse-labeled at three minutes after infection (early mRNA was degraded exponentially), with a half-life of about 2.2 minutes. In contrast, mRNA pulse-labeled at 15 minutes after infection showed more complex decay. During the first four to five minutes, about 25% of the mRNA was degraded relatively slowly. Thereafter the remaining X-specific mRNA was rapidly degraded with a half-life of 1.4 minutes. This characteristic decay pattern of late h mRNA was observed in all of six replicate experiments. Under these conditions, E. coli mRNA decayed exponentially with a half-life of 1.3 minutes (Fig. l), which is similar to that observed for late X mRNA after the initial lag. Degradation of phage mRNA at a rate comparable to or slower than that of host mRNA would not be surprising; however, this is the first case in which an initial slow phase of decay has been observed before the onset of bulk, rapid decay of 15

Time

(b)

(rnln)

Pm. 2. Residual synthesis of RNA at various times after rifampicin addition, and the effect of a cold uridine chase on the incorporation of [sH]uridine. (a) Cells of E. co& W3110 (MS57 87) were heat-induced and after 16 min at 42”C, a sample of the culture was pulse-labeled with [sH]uridine (10 @/ml) for 1 mm. Rifampicin (final concn 300 H/ml) was added to the rest of the culture and at various times portions of the culture were pulse-labeled with [3H]uridine (10 #X/ml) for 1 min. Acid-preoipitable counts were determined in each case. Radioactivity incorporated during a 1-min pulse (before rifampicin addition) was taken as 100% (0 time). (b) [sH]uridine (10 pCi/ml, spec. act. 4.3 Ci/mmol) was added to a culture of E. coli W3110 (hcI857 57) that had been induced for 15 min at 42°C. I.5 min after the addition of [3H]uridine was added to a portion of the culture (0 time), unlabeled uridine (final conen 200 pg/ml) (-•--•--). Incubations were continued, samples withdrawn at various times and acid-precipitable radioactivity determined.

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RNA

mRNA. A trivial explanation of the lag could be that the late X mRNA transcripts are much longer than the early ones (Gariglio & Green, 1973; Chowdhury & Guha, 1973) and in that case, loss of labeled sequences soon after addition of rifampicin would be balanced by the continued incorporation of label into distal sequences. In fact, Green et d. (1970) have been observed that at 20°C transcription of late X mRNA continues for eight to ten minutes after the addition of rifampicin, and correspondingly very long transcripts can be detected (Gariglio & Green, 1973). However, at 42°C where RNA polymerase should move at a rate about five times as fast, synthesis of even the longer transcripts would be expected to finish in 1.5 to two minutes (rather than the 8 to 10 min at 20%). This expectation is confirmed in experiments like that of Figure 2. In this experiment, the residual incorporation of [3H]uridine into nascent RNA was measured at intervals after rifampicin addition. As seen in Figure 2(e), at one and two minutes after the addition of rifampioin (300 pg/ml), the rate of RNA synthesis falls to 8 and 2% of the initial rate. All detectable synthesis of RNA was thus completed within three minutes of rifampicin addition. When 10d3 M-unlabeled uridine was added after the pulse, then even in the absenceof rifampicin, residual incorporation was curtailed even more (Fig. 2(b)). Thus in the experiments of Figures 1, 3 and 4, where both rifampicin and unlabeled uridine were added, the lag in decay of late XmRNA cannot be attributed to a balance of decay and prolonged continued synthesis. 18

Imin

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6min

+

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16s +

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‘;”

Gradient

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fraction

Fm. 3. Sucrose density-gradient analysis of total and late h mRNA. The experiment was done as described in the legend to Fig. 1 and in Materials and Methods. 0.5 ml of late X RNA preparations (purified from cells harvested at 1, 2, 4, 6, 8 and 10 min after rifampioin addition) were gently layered on 17 ml 10% to 30% sucrose gradients (see details in Materials and Methods). After the runs, about 30 fractions were collected from each gradient. Total 3H, W as well as radioactivity hybridizable to /\ DNA, were determined for each fraction. All gradients have been normalized with respect to a constant W radioactivity. --a--@--, total [sH]RNA.; -x-x-, [3H]RNA hybridizable to h DNA.

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4 6 Time(mln)

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FIQ. 4. Size-distribution analyses of pulse-labeled late h mRNA in various size classes at different times after rifampiein treatment. Data for these analyses have been obtained from Fig. 3. hspecific mRNA from each gradient was divided into 5 size classes (I to V). The amount of labeled mRNA in each size class at various times after rifampicin treatment was computed; in each ease the RNA present at 1 min was taken as 100%. Total X mRNA was obtained by summing the subfractions.

(b) Bucroseden&y-gradient analysesof h messengerRNA in the presenceof rifam(picin To understand the events occurring during the lag in decay of late X mRNA, we investigated the size of mRNA at various times after the addition of rifampicin. In an experiment similar to that described in Figure 1, RNA was extracted from cells at 1, 2, 4, 6, 8 and 10 minutes after rifampicin addition and analyzed by sedimentation in a sucrosedensity-gradient. Total C3H]RNA, as well as X-speci& mRNA was determined in each fraction of the gradient (Fig. 3). All the gradients shown in Figure 3 have been normalized to a constant input of 14Ccounts, and the positions of the [14C]rRNA are indicated by the arrows. As seen in Figure 3(a), one minute after rifampicin addition, peaks of h-specific mRNA sedimenting at 30 8, 25 S, 23 S, 14 S and 8 S were seen, The observed sedimentation pattern of h-specific mRNA is similar to that reported in earlier studies (Oda et al., 1969; Kourilsky et al., 1971). The very long transcripts (~60 S) reported by Gariglio & Green (1973) and Chowdhury & Guha (1973) were not observed, and probably are very rapidly processedat 42°C. By four minutes, while there was little loss of total h-specific mRNA, much of the heavier sedimentating (>22 S) mRNA was no longer observed. To analyze these data more quantitatively, the gradients were arbitrarily divided into five regions (I to V, from largest to smallest transcripts; seethe panel of Fig. 3 for the sample harvested 10 min after rifampicin addition). The amount of A-specific mRNA in each region was then computed and plotted as a function of time after rifampicin addition. According to the data in Figure 4, (1) initial relatively slow

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decay was again evident for about four minutes before the bulk of late X mRNA was degraded (dotted curve); and (2) the larger the size class of RNA, the more rapidly it was degraded; (3) as a result, by four minutes, when the fraction of labeled RNA in regions I, II and III had decreased by 80, 55 and 25%, respectively, the fraction in regions IV and V showed an increase of 8 and 65%, respectively.

4. Discussion The results show that in contrast to other bacteriophage mRNA, e.g. T4 (Craig et al., 19’72) +X174 (Hayashi & Hayashi, 1968), X13 (Puga et al., 1973), Ml3 (Jaenisch et al., 1970) and T7 (Summers, 1970), which are relatively resistant to chemical decay, h mRNA is degraded at a rate comparable to that of the host. The data show also that early and late h mRNA have different decay characteristics. Early X mRNA decayed exponentially from the beginning with a half-life of 2.2 minutes, whereas the late mRNA exhibited a lag or slower phaseof decay for about four minutes before the onset of rapid degradation (ta = 1.3 min). The final rate of degradation of late h mRNA was as fast as that of E. coli mRNA-though of coursethe enzymatic mechanism might be quite different. A similar, though shorter lag (3 min) has also been reported recently for ara-specific mRNA (Cleary 82;Englesberg, 1974). The difference in the decay behavior of early and late XmRNA could either be due to some unique property of late X mRNA, or may be conferred by some X function (expressedafter 3 min) acting on late mRNA. In the latter case,study with mutants in early h functions (e.g. b,, tof, etc.) would be extremely useful. However, it also remains to be seen whether newly-formed early mRNA species assayed late in infection exhibit a decay rate characteristic of late mRNA, or continue to decay as they do in the initial minutes of phage development. The size-distribution of pulse-labeled late h mRNA at intervals after rifampicin treatment (Figs 3 and 4) indicated that during the “lag period” considerable and progressive fragmentation of the larger transcripts occurred. The small fragments that increased as a result are apparently too small to code for any normal sized proteins. The physiological basisof the fragmentation and its possiblerelation to the lag in decay are not known. However, at least three possibilities, not mutually exclusive, could be in accord with present thinking: (1) the initial breaks represent processing cleavages (Hercules et al., 1974; Dunn & Studier, 1973; Nikolaev et al., 1973). The messagescould contain sizeable regions that are not translated but are cleaved away from translated segmentsand are then degraded. “Processing” might be independent of decay, or required for translation, subsequent degradation, or both; (2) the endonucleolytic cleavages produce intermediates in normal chemical decay (Blundell & Kennell, 1974; Blundell et al., 1972). Any mRNA, in this case, would then decay by a seriesof random cleavages, followed by degradation of the fragments. In such a model, the original X mRNA speciesare so long that a large number of cleavages are required to reach the point at which acid-soluble fragments (or nucleotides) could be produced rapidly by further endo- or exonuclease action; (3) the cleavagesproduce intermediates thay may represent a slower decay mechauism that is ordinarily masked by the normal mechanismsof decay, or may represent only rare, irrelevant events that occur when an mRNA molecule is stored in a cell. Both these versions of alternative (3) supposethat the cleavages are totally outside the usual mode of mRNA decay.

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In the casesof alternatives (1) and (3) late mRNA would be protected at least transiently by a “stabilizing” factor or influence in the infected cells. In the caseof alternative (2), the simplest inference would be that late X mRNA can only decay by endonucleolytic breaks to ever smaller fragments. Becauseof the progressive breaks the actual kinetics would then be “multi-hit” (rather than biphasic) whereas the early mRNA would be immediately subject to apparent degradation without initial cleavages. The late X transcript would then provide the first clear caseof progressive or multi-step decay, with an apparent lag or “slow phase” before rapid degradation ensues.

While alternative (2) seemsplausible and simple, the comparison of the fate of late h mRNA and p,-promoted trp mRNA (Yamamoto & Imamoto, 1975) weakens it considerably. In both cases,transcripts show some endonucleolytic breaks during a period when little bulk degradation is observed; but while rapid exponential decay of late X mRNA is finally observed, p,-trp mRNA remains “stable”, including the long trp sequencethat requires no multi-hit preparation for decay. Thus, it seems premature to favor one or another alternative, even speculatively. Studies are grateful

were supported to Dr F. Imamoto

by grant for

GB23052 from helpful discussions

the National and

Science Foundation.

communication

of recent

We results.

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70, 2171-2175. Silengo, L., Nikolaev, N., Schlessinger, D. & Imamoto, F. (1974). 7-19. Skalka, A. (1966). Proc. Nat. Acad. Sri., U.S.A. 55, 1190-1195. Summers, W. C. (1970). J. MoZ. BioZ. 51, 671-678. Yamamoto, T. & Imamoto, F. (1975). J. Mol. BioZ. 92, 289-309.

Mol.

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Tenet.,

134,