Correlation between RNA Synthesis and Basal Level Guanosine 5

THEJOURNAL

OF BIOLOGICAL CHEMISTRY Vol. 256. No. 22, Issue of November 25, pp. 11651-11656, 1981 Printed in U.S.A.

Correlation between RNA Synthesis and Basal Level Guanosine 5’Diphosphate 3”Diphosphate in Relaxed Mutants of Escherichia coZi* (Received for publication, March 23, 1981)

Peter A. Lagosky$ and F. N. Chang From the Department of Biology, Temple University, Philadelphia, Pennsylvania19122

Through the useof a new nucleotide extraction pro- bacteria (14-16), but until recently, this has not been the case for relaxed mutants (17). The ability to recover substantially cedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response toamino more ppGpp from relaxed mutants during balanced growth acid starvation (Lagosky, P. A., and Chang, F. N. (1980) than hadbeen previously reported in these strains has enabled J. BacterioL 144,499-508). The basal level amounts of us to demonstrate that both reM mutants and phenotypically guanosine5”diphosphate3”diphosphate(ppGpp)in relaxed reM’ rplK strains exhibit a previously undescribed both relA and phenotypically relaxedrelA+ rplK (relc) response to amino acid limitation (17). These studiesshowed strains were shown to decrease at the onsetof amino that at theonset of amino acid starvation, relA SPOT’,reZA acidlimitationandtoremainseverelydepressed SPOT,and reM+ rplK SPOT+mutants display an immediate throughout the course of the starvation. Upon resup- and sustained decline in their ppGpp content to amounts plementation of amino acid-starved relaxed mutants, much less than their original basal levels (17). These diminthe production of ppGpp resumes and results in the ished levels of ppGpp are maintained throughout the course of this nucleotide beyond temporary overaccumulation of the starvation, increase rapidly upon addition of the reits original basal level amount.We now show that the basal level ppGpp content of relaxed bacteria, as well quired amino acid, and temporarily exceed their prestarvation as its subsequent fluctuations in response to amino basal acid level amounts before eventually returning to their preexisting basal levels (17).A rough inverse correlation between starvation, is inversely correlated with the initial rates of RNA synthesis in these strains. The ability of ppGpp RNA accumulation and the ppGpp level during amino acid to control the rateof protein synthesis in relA mutants starvation and subsequent resupplementation has also been was also examined. It was observed ppGpp that hadno observed in these relaxed mutants (17). In the present report, apparent direct effect on the initial rates of protein we have investigated the above correlation further and found that a precise inverse coupling exists between cellular ppGpp synthesis in relA mutants. Theconstantinversecorrelationwhich exists be- levels and initial rates of RNA synthesis. We have also chartween ppGpp content in relA mutants, and their rates acterized the ribosomal RNA species that are overproduced of RNA synthesis provide evidence which indicates that during amino acid starvation and studied theirfateafter basal level ppGpp synthesis has definite physiological amino acid resupplementation. The significance of these findsignificance. Italso suggests that the synthesis of basalings are discussed in relation to the role of ppGpp as a level ppGpp might be an absolute requirement needed modulator of RNA synthesis during all phases of cell growth. for normal bacterial growth. EXPERIMENTALPROCEDURES

The ability of wild type bacteria to reduce their production of stable RNA in response to amino acid limitation has been linked to their capacity to synthesize the unique nucleotide guanosine 5”diphosphate 3”diphosphate (1-4). Although reM mutants do not produce ppGpp when subjected to amino acid starvation, they do maintain basal levels of this nucleotide during balanced growth (5-7)and, like stringent bacteria, they can adjust their intracellular content of ppGpp in response to various other physiological conditions (5,6, 8-10). Studies conducted with reM’ and relA strains containing spoT mutations have shown that thebasal level of ppGpp which a cell maintains directly influences both its metabolic activity and its growth behavior (2-4, 8, 11-13). An inverse correlation between RNA synthesis and the ppGpp level of the cell has been well documented in stringent * This investigation was supported in part by Public Health Services research Grant GM-19302 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address, Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

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Materials-Carrier-free orthophosphoric acid HtZP04,[’4C]uracil, [14C]arginine, and Formula 949 scintillation fluid were obtained from New England Nuclear Co., Boston, MA. Polyethyleneimine cellulose thin layer chromatography sheets (Polygram Cel 300 - precoated plastic sheets, 20 X 20 cm) were purchased from Brinkmann Instruments, Inc., Westbury, NY. Radioautography and fluorography were carried out with Kodak x-ray film (X-Omat R).Nonradioactive nucleoside phosphates were obtained from P-L Biochemicals, Inc., Milwaukee, WI. All other reagents were of analytical grade and were obtained from local sources. Bacterial Strains-The following Escherichia coli K12 strains were used in this study: KL99 (Hfr P042, thi-1,r e a l , spoTl,lac-42) (18), NF161 (F-, metB1, argA52, rea’, spoT1) (19), NF859 (F-, metB1, argA52, relA+, SPOT’) (19), and PLlO (F-,metB1, relA1, SPOT’) (17). PLlOwas derived from an interrupted Hfr mating between KL99 and a spontaneous streptomycin resistant mutant of NF859. It carries the reL.41 marker from KL99 and is streptomycinresistant. Media, Growth Conditions, and Radioactive Labeling-All bacterial strains were grown at 37 “C in Hershey’s Tris minimal medium (20) containing 0.33 m~ phosphate, 0.3% glucose, thiamin (2 pg/ml), and 50 pg/ml of each required amino acid or nucleotide. Amino acid starvation was initiated by the addition of 500 pg/ml of valine and was reversed by adding 500 pg/ml of isoleucine. RNA accumulation was measured by labeling cells with carrierfree H332POs(1.5 pCi/ml) which was added two generations prior to sampling. DNA content was also assayed and as indicated below, the

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RNA Synthesis and Basal Level ppGpp

160 pgof unlabeled carrier E. coli tRNA. Millipore filtration and determination of radioactivity were the same as stated previously. The initial rates of RNA synthesis were expressed as the counts per min of [I4C]uracil incorporated into cold acid-insoluble material per min of exposure to theradioactive pulse per A720. Measurement of the Rate of Protein Synthesis-The initial rates of protein synthesis were measured by pulsing individual 0.5-ml aliquots of unlabeled culture with 1.5 pCieach of [I4C]arginine(0.203 Ci/mol of arginine) for 4 min (23). Labeling was stopped by the addition of ice-cold 20% trichloroacetic acid and all samples were subsequently boiled at 100 “C in a water bath for 20 min. Millipore filtration and quantitation were the same as for the RNA samples measured above. The initial rates of protein synthesis were expressed as the counts per min of [I4C]arginineincorporated into hot trichloroacetic acid-insoluble material per min of exposure to the radioactive pulse per A7m. Extraction of H332P04-labeledRNA for Gel ElectrophoresisHPP04-labeled RNA samples for use in gel electrophoresis were each derived from the cell pellets of 5 ml of whole culture. All samples were extracted as detailed by Lindahl(24)except the sucrose gradient fractionation step was eliminated. After DNase treatment, the whole cell extracts were used for gel electrophoresis without further purification. RNA Gel Ele~trophoresis-H~~PO~-labeled RNA whole cell extracts were analyzed by electrophoresis on 3%polyacrylamide, 0.3% agarose composite gels followingthe procedure of Peacock and Dingman (25). These gels were prepared as vertical slabs (20 cm X 20 cm X 3 mm) with 20 sample slots (24). Ten of each whole cell H?2P04labeled RNA extract were underlaid per slot and were run at 9 V/cm at 4 “C. With this procedure, small RNA molecules are lost, but 17.5 S and 16 S rRNA are clearly separated (24). The gels were subsequently fixed, stained (25), and dried onto Whatman No. 3 paper. Following radioautography, the radioactive areas of the dried gel were then cut out and counted in 5 ml of Bray’s Solution (26). All samples were quantitated as nanomoles of phosphate per A7m. RESULTS

Correlation between ppGpp Synthesis and RNAAccumulation-Using an extraction procedure which recovers approximately 4 times more ppGpp from the cell thanthe conventional formicacid method (21), we had previously shown that the basal levels of ppGpp in all relaxed strains of E. coli decreased in response to amino acid starvation (17). A rough inverse correlation between RNA accumulation and the ppGpp level during amino acid starvation and subsequent resupplementation had also been observed in these relaxed mutants (17). In our previous studies, however, RNA accumulation was quantitated asradioactive counts per min using [14C]uracilwhile ppGpp wasassayed in separate cultures labeled with H332P04(17). To more accurately test the relationship between ppGpp content during and after amino acid starvation and its influence on RNA accumulation, both parameters were measured in the same H332P04-labeledculture

parent unresponsiveness of the rateof accumulation of RNA to a large change in the ppGpp level followingthe fist20 min of resupplementation (41 to 60 min), these results appear to indicate that overall RNA accumulation in KL99, during its recovery from amino acid starvation, is inverselyrelated to its ppGpp content (see below). Influence ofppGpp on the Initial Rates of RNASynthesis in KL99”Since the rateof RNA accumulation represents the difference between the rateof RNA synthesis and the rate of its degradation (10, 27), the initial rates of RNA synthesis in KL99 were examined during balanced growth as well as in

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FIG.1. Correlation of ppGpp synthesis with RNA accumulation in KL99. One culture of KL99 (relAI spoT1) was grown in Hershey’s Tris minimal medium with 0.33 m~ phosphate and was labeled with H332P04(1.5 pCi/ml) which was added two generations before the first sample was taken. At -10 min, the culture was split and 1part was maintained as an unstarved control. Five hundred pg/ ml of valine was added to the other part at time zero and 500 pg/ml of isoleucine was added at 41 min. Samples for nucleotide content and RNA production were taken from the same culture flask and were processed according to procedures outlined under “Experimental Procedures.” The RNA measurements were corrected for DNA content by subtracting alkaline-resistant trichloroacetic acid-precipitable radioactive counts from duplicate culture samples as described under “Experimental Procedures.” Unstarved (0);starved and resupplemented (a).

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FIG. 2. Influence of ppGpp production on the initial rate of RNA synthesis inKL99. A cultureof KL99 was grown in Hershey's Tris minimal medium containing 0.33 m~ phosphate and was split into 2 equal volumes at time -20 min. One portion was saved as an unstarved control and the other was subjected to amino acid starvation at time zero by the addition of 500 pg/ml of valine. Starvation was subsequently reversed by the addition of 500 p g / d of isoleucine at theindicated time. Samples for determination of the initial rate of RNA synthesis were removed from the culture flask and pipetted into tubes containing ['4C]uracil (5 pCi/87.5 pmol/ml) and 10 times this concentration of unlabeled cytidine and were allowed to incubate for 4 min a t 37 "C before stopping the growth by adding 2 volumes of icecold 20% trichloroacetic acid and blending vigorously on a Vortex mixer. AU samples were then processed as outlined under "Experimental Procedures." The ppGpp profde contained in this figure was obtained from the experiment shown in Fig. 1 (toppanel).

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FIG. 3. Fate of the RNA produced in KL99 during amino acid starvation. Growth conditions and H2'P04 labeling were the same

as in Fig. 1. At -10 min, the culture was split and 1 part was maintained as an unstarved control. Amino acid starvation and resupplementation were carried out as in Fig. 1. The RNA in these extracts was then separated electrophoretically on 3% acrylamide, 0.3%agarose composite slab gels. Solid figures represent unstarved and open figures represent starved and resupplemented samples. 23 S and larger (A, A); 17.5 S (0,O); 16 S (0,

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the initial rates of RNA synthesis demonstrated in reZA mut a n t s , however, provides new evidence to substantiate therole of ppGpp as a control element of transcription and suggests that ppGpp uniformly serves as amodulator of RNA synthesis during balanced growth as well as under nutritionally unfaresponse to amino acid starvation and subsequent resupple- vorable conditions. Characterization of RNA Produced during Amino Acid mentation in order to clarify the apparent discrepancy mentioned in the previous section. The results presented in Fig. 2 Starvation in relA Mutants-The nature of the RNA proindicate that a 90% decline in basal level ppGpp content duced in KL99 during amino acid starvation and its fate as a occurs during the first 20 min of starvation and that this result of resupplementation of starved cultures was examined corresponds to approximately a 60%increase in the initial rate electrophoretically on 3%polyacrylamide, 0.3%agarose comof RNA synthesis in this strain. The ppGpp level remains posite slab gels(24,25).WhenH;'PO4-labeledwholecell severely depressed throughout the remainder of the starvation extracts wereused asthe RNA source large increases in period with almost no additional change between 20 and 40 precursor ribosomal RNA components (23 S and larger' and min. This is coordinately reflected in only a very slight addi- 17.5 S) were noted immediately after starvation commenced tional increase in the level of the initial rates of RNA synthe- (Fig. 3). To show that the increase in the precursor rRNA in sis. It should be noted that thedrop in ppGpp level precedes the cell extracts ofKL99 is not due to an artifact of the the increase in the rateof RNA synthesis. For example, 2 min extraction procedure, whole cell RNAextracts of PLlO (relAl after the addition of valine, the ppGpp level had dropped 50% SPOT+), NF859 (reZA' SPOT+), and NF161 (relAfspoT1) were whereas only a small increase in the rate of RNA synthesis also subjected to polyacrylamide-agaroseslab gel electrophowas detected. resis under the same experimental conditions. The results Immediately after resupplementation of the amino acid- presented in Fig. 4 (top panel)indicate that the behavior of starved KL99 culture with isoleucine, the ppGpp content PLlO issimilar to theresponse detailed above for KL99 except begins to increase and within the next 20 min it accumulates that themagnitude of the changes in its rRNA levels is not as to an amount which is equivalent to its original prestarved great (as predicted from the fluctuations in the total RNA basal level. During this same 20-min time span (41 to 60 min), content of this strainduring amino acid starvation (17)).More the rate of RNA synthesis progressively declines until it too importantly, however, the results with NF859 and NF161 (Fig. reaches its prestarvation rate. The succeeding 30 min show 4, middle and bottom panel, respectively) are consistent with the characteristic overaccumulation of ppGpp to an amount the response of stringent bacteria to amino acid starvation which is about twice the basal level present during balanced reported in the literature (4, 11, 15, 16). (i.e. no net accumugrowth and thiscorresponds to a40% decrease from the basal lation of rRNA during the period of starvation) indicating level rate of RNA synthesis. As the ppGpp content gradually that theaccumulation of radioactivity in the precursor rRNA returns to its prestarvation basal level, the initial rates of areas of the slab gels fromKL99 and PLlO whole cellextracts RNA synthesis again increase until they too attain a level during amino acid starvation is not an artifact of the extraction which is characteristic of balanced growth for this strain. This procedure. precise inverse coupling between ppGpp contentand the Fate of the RNA Produced in Relaxed Mutants during initial rates of RNA synthesis suggests that ppGpp may con- Amino Acid Staruatwn-Polyacrylamide-agarose gel electrotrol the accumulation of RNA in relaxed mutants in response phoresis of amino acid-starved whole cell extracts from reZA to amino acid starvation andsubsequent recovery through its mutants indicates that the content of precursor ribosomal action on the rate of RNA synthesis. In the first 20 min RNA begins to increase at the onset of starvation and that following resupplementation (41 to 60 min), there is adiffer- this accumulation persists throughout the course of the amino ence in the responsiveness to changing levels in ppGpp beI Technical difficulties with this type of gel electrophoresis system tween the initial rates of RNA synthesis and the ratesof RNA do not permit accurate reproducible separation of extremely large accumulation. This difference could becaused by a change in RNA molecules derived from unfractionated cell extracts so all radiothe rateof RNA degradation (which was not measured) during active molecules larger than 23 S were always quantitated as a single this period of time. The correlation between ppGpp levels and unit.


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starvation of KL99 (Fig. 1, bottom panel) as well as the elevated levels of both mature 23 S rRNA and 16 S rRNA which result from its subsequent processing eventually decline until they reach their prestarvation basal levels. These results indicate that after processing the excess RNA which accumulated during amino acid starvation, relaxed bacteria degrade it and therefore are not likely to significantly increase their ribosome content beyond the amount dictated by their current growth potential. The Influence of ppGpp on the Initial Rates of Protein Synthesis in KL99-The maintenance of normal translational fidelity in relA' bacteria during periods of limited availability of a specific aminoacyl-tRNA species has been attributed to the large increase in ppGpp content which accompanies amino acid starvation (23). The hypothesis which has been put forward to explain these results rests on the premise that translational activity is directly influenced by ppGpp such 235 8Lorpr that when the ppGpp levelof a culture is increased either the NF859 rate of binding the cognate aminoacyl-tRNA-EF-Tu. GTP ternary complex to theA-site of the ribosome (28) or the rate of peptide chain elongation (29) decreases. The simple prediction made by this hypothesis is that a direct inverse relationship exists between ppGpp content and the rate of protein biosynthesis. The results used to substantiatethis claim, however, wereobtained solely through comparative studies of I + Ins 4 I I relA' bacteria and relA mutants during amino acid starvation 0 15 30 45 60 75 (23). MINUTES The discovery that relA spoT mutants accumulate twice as much ppGpp during recovery from amino acid limitation as they produce during balanced growth and that this increase can reduce the initial rates of RNA synthesis in these strains NF161 by 60% revealed the existence of an unique situation in which the cell has the capability of synthesizing protein at the same time that it is accumulating substantial amounts of ppGpp. 23s 8 Larger g" 200 The influence of ppGpp content on the initial rates of protein synthesis in KL99 was therefore examined during this period in order to test thehypothesized inverse relationship between these two parameters under non-starvation conditions. The results presented in Fig. 5 indicate that, within 3 min of the onset of amino acid starvation, the rateof protein synthesis in this strain decreased to alevel which represents a 70%reduc0 15 30 45 60 75 tion from its rate during logarithmic growth and which it MINUTES maintains throughout the entire period of starvation. The FIG. 4. Electrophoreticcharacterization of whole cell-ex- synthesis of basal level ppGpp also drops in response to amino tracted RNA produced in PL10, NFSS9, and NF161. Growth conditions and H332P04labeling were the same as in Fig. 1. All acid starvation as demonstrated previously. Immediately after isoleucine has been added back to the cultures andsamples were treated as in Fig. 3. SoZid figures represent unstarved and open figures represent starved and resupplemented isoleucine-starved culture, the initial rate of protein synthesis samples from PLlO (reZAl SPOT'),NF859 (reZA' spoT'), and NF161 increases dramatically, and within 3 min, it shows a &fold (reZA+spoT1). 23 S and larger (A, A); 17.5 S (0,0 ) ;16 S (0,W). increase over its residual starvation rate (representing a 60% increase from its prestarvation rate). Thisis not anunexpected acid limitation (Fig. 3; Fig. 4, top panel). Fig. 3 also reveals that the precursor rRNA molecules contained in the 17.5 S fraction are apparentlyprocessed into mature 16 S ribosomal RNA species after a time lag of about 5 min following resupplementation. These results also indicate that there is no apparent change in the 23 S and larger RNA fraction (which contains 30 S and 25 S precursor rRNA in addition to 23 S rRNA) during this same period of time. The apparent maintenance of the elevated levels of 23 S and larger rRNA found in the whole cell extracts after amino acid resupplementation (Fig. 3), however, can be explainedas arising fromthe continued, although progressively diminished, elevation in the level of the rates of RNA synthesis (Fig. 2). This would result in the continued accumulation of total cellular RNA in excess of FIG. 5. Influence of amino acid starvationon the initial rate the basal level amount for the initial 20-min period following of protein synthesis in KL99. This experiment was performed isoleucine addition (between 40 and 60 min). The increased exactly as outlined in Fig. 2 except no cytidine was added and ["C] amounts of total cellular RNA accumulated during amino acid arginine (6 pCV83.0 pmol/ml) was used instead of ['4C]uracil.

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extended periods of time provides new evidence to support the hypothesis that ppGpp is the control element of RNA synthesis during periods of metabolic imbalance (3, 4, 8, 10, as well as during balanced growth (5, 7 ) . 11,15,16,32-35) (Recently, E . coli mutants (reZS),which lack basal level ppGpp, have been reported (36). The ppGpp levels in these strains were determined using the conventional formic acid extraction procedure which is known to cause ppGpp degradation (21).We have observed that relS mutantsdo maintain small but measurable basal levels of ppGpp when this nucleotide is extracted by the lysozyme-deoxycholate procedure.') A Model for the Involvement of ppGpp as a Control Element during All Phases of Cell Growth-The inverse correlation between the ppGpp content of relA mutants and their rates of RNA synthesis suggest the possible existence of a mechanism for the autogenous regulation of RNA production DISCUSSION in all strains of bacteria which produce ppGpp. As stated Tbe ability of relaxed bacteria to greatly reduce their syn- previously, the decline of the ppGpp basal level of a relaxed thesis of basal level ppGpp in response to amino acid starva- mutant in response to amino acid starvation causes an increase tion has provided an opportunity to re-examine the influence in its rate of RNA production. Due to a lack of sufficient of ppGpp synthesis on RNA production. The reductions in protein synthesis (Fig. 5) (4,23,37-39),however, the majority basal level ppGpp which accompany amino acid starvation of of the RNA which is produced during this period cannot be relaxed bacteria were found to be coordinated with increases immediately utilized and therefore begins to accumulate (Figs. in boththeir initial rates of RNA synthesis (Fig. 2) and, 3 and 4) (3, 4, 11, 15, 33). When the required amino acid is consequently, with total RNA accumulation (Fig. 1). Slab gel added back to a starved culture the capacity to synthesize electrophoresis performed on RNA extracts of relaxed bacte- protein returns (Fig. 5 ) and is accompanied by the resumption ria during amino acid starvation indicate that a large majority of ppGpp synthesis on the ribosome (7). The paradox which of the RNA which accumulates at this time is in the form of immediately presents itself, however, is that during this period precursor ribosomal RNA species (Figs. 3 and 4) (16, 24, 30, of recovery from amino acid starvation, while the culture is 31). The fact that these precursor ribosomal RNA molecules growing, it is accumulating large amounts of ppGpp and are apparently stable throughout the course of the starvation gradually reducing its synthesis ofnew RNA (Fig. 2). (This period indicates that proper rRNA processing requires contin- situation had not been broached previously in the literature ued protein synthesis (30, 31) and suggests that the cell does due to the lack of information about the response of basal not randomly destroy potentially useful RNA species during level ppGpp synthesis in amino acid-starved relA mutants.) periods of metabolic imbalance. Following resupplementation The simplest way to account for this would be to postulate a with the required amino acid, however, relaxed cells begin to mechanism which would be sensitive to some consequence of process the accumulated precursor rRNA species (Fig. 3). excess RNA production and which could interact with the During this same period of time (41 to 60 min), the levels of gene product of the spoT locus causing it to shut off degrappGpp in these strains begin to increase, causing gradual dation of basal level ppGpp in a manner similar to theresponse reductions in the accumulation of new RNA due to a decrease observed when both stringent and relaxed SPOT' and spoT in the rate of RNA synthesis. The consequence of processing strains are subjected to a carbon source downshift ( 7 ) . This and subsequent degradation of accumulated rRNA precursor would allow ppGpp to accumulate through normal basal level molecules (Fig. 3) and the reduced accumulation of new RNA production and, at the same time, would not interfere with (Fig. 1) is reflected in a net loss of RNA from the bacterial protein synthesis (Fig. 5). The cell would therefore have the culture (60 to 90 min) until the prestarved basal level content ability to determine the amount of RNA which is contained of RNA is reached. These results indicate that relaxed bacteria and to modulate its rate of RNA synthesis until it could are not likely to increase their ribosome number beyond some properly dispose of any excess. This proposed mechanism may limit dictated by their current growth potential. also be operative in unstarved bacteria (40,41). The eventual overaccumulation of ppGpp in relaxedstrains The fact that relA mutants must be used to demonstrate during recovery from amino acid starvation was found to be the possible existence of this mechanism should not be interaccompanied by a temporary decline in the rate of RNA preted asan indication that they have evolved a special synthesis below the level characteristic of balanced growth physiology distinct from wild type bacterium. The relA locus (Fig. 2). Conversely, following a transient surge in the initial controls the production of stringent factor (34, 42-45) which rates of protein synthesis, the prestarvation rate of poly- is a component of this same control mechanism. The ability peptide production was resumed while the ppGpp content was to observe the functions of this postulated mechanism in a still much higher than its basal level (Fig. 5; 70 to 120 min). relA background results solelyfrom the capacity of these The fluctuations in the initial rates of RNA synthesis, how- mutants to survive after removal of this component of the ever, were found to be inversely correlated with the ppGpp system. content of the culture throughout the entire period of logaSeveral attempts were made3to verify the presence of ppGp rithmic growth, amino acid starvation, and recovery following (MS 111) (46) in the nucleotide extracts used to quantitate the resupplementation (Fig. 2). It appears, therefore, that the ppGpp measured during the course of this study.We were not accumulation of excess RNA which occurs during starvation able to reproducibly recover measurable amounts of ppGp and the modulation of protein synthesis which accompanies from relA mutants using the lysozyme-deoxycholate extracthis response both result from changes in the rate of RNA tion procedure (21) and under no circumstance was a comesynthesis brought about by fluctuations in the ppGpp content of the cell. This inverse relationship between ppGpp levels ' P. A. Lagosky, unpublished work. and the initial rates of RNA synthesis in relaxed bacteria over C. C. Pao, personal communication.

result in as much as a significant proportion of the RNA whichwas synthesized during starvationmust have been messenger RNA. This large increase in the rate of protein synthesis is maintained for about 25 min (41 to 65 min) and corresponds approximately to the time period required for ppGpp to accumulate toits prestarvation basal amount. Within the next 60 min, however, whilethe ppGpp content of the culture is at its highest, the initial rates of protein synthesis return to the level of their prestarvation rate and maintain this position despite an initial doubling of the basal amount of ppGpp followed by a 50% decline in its content. The lack of correlation between ppGpp content and the initial rates of protein synthesis revealed by these results indicates that ppGpp cannot be directly involved in the control of the rate of protein synthesis.

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lation between ppGp content and the initial rates of RNA synthesis observed in these strains.’ In order to obtain measurable amounts of ppGp, the lysozyme nucleotide extracts required re-extraction with formic acid. This also resulted, however, in an almost complete disappearance of ppGpp from tbe resulting extracts (17, 21).2 The closely coupled inverse correlation between ppGpp content and the initial rates of RNA synthesis demonstrated in this report, together with the above-mentioned inconclusive results, argues strongly in favor of a direct role for ppGpp in the control of RNA synthesis. REFERENCES 1. Cashel, M., and Gallant, J. (1969) Nature 221, 838-841 2. Fiil, N. P., von Meyenburg, K., and Friesen, J. D. (1972) J . Mol. Biol. 71, 769-783 3. Khan, S. R., and Yamazaki, H. (1974) Biochemistry 13, 27852788 4. Sokawa, Y.,Sokawa, J., and Kaziro, Y. (1975) Cell 5, 69-74 5. Friesen, J. A., Fiil, N. P., and von Meyenburg, K. (1975) J. Bwl. Chem. 250,304-309 6. Gallant, J. D., Erlich, H., Hall, B., and Laffler, T.(1970) Cold Spring Harbor Symp. Quant. Biol.35,397-405 7. Gallant, J., Margason, G., and Finch, B. (1972) J. Biol. Chem. 247,6055-6058 8. Chaloner-Larsson, G., and Yamazaki, H. (1978) Can. J. Biochem. 56,264-272 9. Lazzarini, R.A,, Cashel, M., and Gallant, J. (1971) J. Biol. Chem. 246,4381-4385 10. Molin, S., von Meyenburg, K., Maaloe, O., Hansen, M. T., and Pato, M. L. (1977) J . Bacteriol. 131,7-17 11. Chaloner-Larsson, G., and Yamazaki, H. (1976) Can. J. Biochem. 54,291-295 12. Chaloner-Larsson, G., and Yamazaki, H. (1976) Can. J. Biochern. 54,935-940 13. Laffler, T., and Gallant, J. (1974) Cell 1,27-30 14. Lazzarini, R.A., and Dahlberg, A. E. (1971) J. Biol. Chem. 246, 420-429 15. Stamato, T. D., and Pettijohn, D. E. (1971) Nature (New Biol.) 234,99-102 16. Gallant, J., and Lazzarini, R. (1976) in Protein Synthesis (McConkey, E., ed) Vol. 2, pp. 309-354, Marcel Dekker, Inc., New York 17. Lagosky, P. A,, and Chang, F. N. (1980) J. Bacteriol. 144, 499508 18. Bachmann, B. (1972) Bacteriol. Rev. 36,525-557 19. Fiil, N. P., Willumsen, B. M., Friesen, J. D., and von Meyenberg, K. (1977) Mol. Gen. Genet. 150,87-101

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