JOURNAL OF BACTERIOLOGY, Jan. 1981, p. 410-4160021-9193/81/010410-07$02.00/0

Vol. 145, No. 1

In Vivo Role of the relA + Gene in Regulation of the lacOperon

PAUL PRIMAKOFFtDepartment ofMicrobiology and Molecular Genetics, Harvard Medical School, Boston,

Massachusetts 02115

Under conditions of amino acid limitation, ,B-galactosidase was produced at a70-fold higher rate in a relAU strain than in an isogenic relA strain of Escherichiacoli K-12. Under identical conditions with the reLA+ and reLA strains carryingvarious lac promoter mutations, rates of 8-galactosidase synthesis in relA+(relative to reUA) ranged from 26-fold higher (promoter mutant P'13) to only 5-fold higher (promoter mutant PrL8uv5). This promoter specificity was independ-ent ofstrain background and the means of eliciting amino acid limitation. Additionof cyclic AMP to the growth medium altered the reLA+/reLA difference for ,8-galactosidase synthesis from the wild-type lac promoter. The experiments suggestthat the reUA/reLA difference in lac expression arises primarily at the point oftranscription initiation. The results are discussed in relation to recent in vitrodata showing a promoter-specific guanosine 5'-diphosphate 3'-diphosphate stim-ulation of lac transcription (P. Primakoff and S. W. Artz, Proc. Natl. Acad. Sci.U.S.A. 76:1726-1730).

Stringent control is a regulatory system whichinhibits the synthesis of stable RNA duringamino acid starvation of Escherichia coli andother bacteria (8). Considerable evidence indi-cates that the nucleotide guanosine 5'-diphos-phate 3'-diphosphate (ppGpp) is directly in-volved in this inhibition (5). In a stringent(reUA+) strain, ppGpp levels increase immedi-ately with the onset of amino acid starvation,and stable RNA synthesis subsequently stops; ina relaxed (relA) strain, ppGpp levels fail to in-crease, and stable RNA synthesis continues (4).This ppGpp-mediated control system has beenthought to serve the function of adjusting syn-thesis rates of tRNA, rRNA and ribosomal pro-tein to the overall rate of cellular protein syn-thesis (5, 7).

Several observations have suggested thatppGpp levels might also affect expression ofgenes in the lac operon. deCrombrugghe et al.(6), Yang et al. (34), and Reiness et al. (27)reported that ppGpp produced a small (usuallytwofold) but reproducible stimulation of 8-galac-tosidase synthesis in an in vitro protein synthesissystem utilizing E. coli S-30. Also, in mutantstrain E25 isolated as thernosensitive for thesynthesis of both tRNA and rRNA (28), highlevels of ppGpp accumulated at the nonpermis-sive temperature, 420C (P. Schedl, Ph.D. disser-tation, Stanford University, Stanford, California,

t Present address: Laboratory ofHuman Reproduction andReproductive Biology, Harvard Medical School, 45 ShattuckStreet, Boston, MA 02115.

1975). At 420C, this strain synthesized f8-galac-tosidase at an elevated rate, as compared withthat of its wild-type parent (Schedl, Ph.D. dis-sertation). Schedl suggested that, at the nnper-missive temperature, the inability of strain E25to make tRNA and rRNA and its capacity tosynthesize excess ,8-galactosidase could be com-mon regulatory consequences of the elevatedlevels of ppGpp in this strain (Schedl, Ph.D.dissertation). Finally, in recent experiments uti-lizing an in vitro protein synthesis system pre-pared from Salmonella typhimurium (2), theaddition of ppGpp resulted in a 20-fold stimula-tion of ,B-galactosidase synthesis (25).

In the present investigation, I have examinedfurther the in vivo relationship of high ppGpplevels to lac expression, by using reLAU/reLAisogenic pairs and some of the large variety ofavailable regulatory mutants of the lac operon.

MATERIALS AND METHODS

Chemicals and media. Minimal medium M-63and rich medium LB have been described previously(22). Isopropyl-/?-D-thiogalactoside (IPTG) was ob-tained from Schwarz/Mann and was used at 1 mM.Cyclic AMP (cAMP) was obtained from Sigma Chem-ical Co.

Bacterial strains and strain construction. Con-struction of strains was accomplished by standardtechniques (22) with the strains listed in Table 1.UL120, UL121, UL128, and UL129 (see Table 4) wereconstructed by mating the A(lac-pro) strain CA7092.1with rpsL derivatives of the isogenic reLA+/reU pair

410

ROLE OF GENE relA+ IN lac OPERON REGULATION 411

of NP290292 and NP290291. Strr Lac recombinantswere picked and mated with E5014.1 and E8027 tointroduce F' lacP+ proA+B+ and F' lacPrL8uv5proA+B+, respectively. The strains denoted Q2through Q9 and Q12 through Q15 (see Table 3) wereconstructed, by the same steps, from the isogenicreU+/reU pair of PP116 and PP117 (Table 1), whichare derived from BF265 (26).

f8-Galactosidase and cAMP assays. The,f-galac-tosidase assay was performed as described previously(22). cAMP levels were measured by the method ofEpstein and co-workers (10, 17).

RESULTS

Synthesis of ,8-galactosidase duringamino acid limitation. reUA+ and relA strainssynthesize B-galactosidase at the same rate insteady-state growth (16), in which both the wild-type and mutant ppGpp concentrations are lowand essentially equal (4). To examine the in vivoeffects of high ppGpp levels on lac enzyme syn-thesis, I induced the operon under conditions ofamino acid limitation. A convenient way toachieve strong amino acid limitation in E. coliK-12 is to add 100 Mg of valine per ml to themedium; adding valine results in a severe, butincomplete, inhibition of isoleucine biosynthesis(21). The experimental design used with valineaddition is to have a relA /relA pair of strainsin steady-state growth in the absence of lacinducer, 10 minutes before induction, valine isadded to give amino acid limitation conditions,and at time zero, IPTG is added to induce thelac operon. Strains Q2 and Q3 are a relA/relApair which carry the lac operon with the wild-type promoter, denoted as P+. After valine ad-dition and lac operon induction in these strains,the synthesis of B-galactosidase at a rate aboveuninduced levels began after a lag of roughly 10min and exhibited kinetics showing excellentlinearity with time for at least 1 h in both reiand relA (Fig. 1A). Under the amino acid-limit-ing conditions, the relA strain Q2 produced f8-galactosidase at a rate 70 times greater than thatof the relA mutant Q3 (Fig. 1A; see Table 3).A potential source of the elevated rate of,-

galactosidase synthesis in relA + is a higher in-tracellular level ofcAMP in relA + during aminoacid limitation. The intracellular cAMP concen-tration was measured and was not significantlydifferent in Q2 relA + and Q3 relA after valineaddition (Table 2). I assayed cAMP levels incollaboration with W. Epstein at the Universityof Chicago. In steady-state growth of Q2 and Q3on glucose, there was a rather low intracellularcAMP level (0.56 ± 0.22 uM; Table 2), whichagrees with results in other E. coli strains (10).cAMP determinations in Q2 and Q3 after valineaddition give essentially identical values for the

TABLE 1. Bacterial strains used for strainconstructiona

Source or refer-Strain Pertinent characteristic ence

NP290291 F- vaiS(Ts) relA lacZ+ F. NeidhardtproA+B+

NP290292 F- valS(Ts) reUA+ F. NeidhardtlacZ+ proA+B +

PP116 W3110 F- lacZ+ P. PrimakoffproA+B+ relA+ rpsL

PP117 W3110 F- lacZ+ P. PrimakoffproA+B+ reLA rpsL

CA7092.1 HfrH A(lac-proAB) 18E5014.1 F' lacP+ proA+B+ 1E8027 F' lacPrL8uv5 1

proA +B+CA8020 F' lacP'laproA+B+ 1CA8023 F' lacPr9proA+B+ 1CA8024 F' lacP'12proA+B+ 1CA8026 F' lacPr13proA+B+ 1

a Strr Lac recombinants were picked and matedwith E5014.1, E8027, CA8020, CA8023, CA8024, andCA8026 to introduce the various F' lac+ proA+B+episomes.

two strains: Q2 relA+, 0.39 + 0.18 ,uM; and Q3relA, 0.34 ± 0.16 ,uM (Table 2).To examine other possible reasons why the

relA + strain can make 70-fold more ft-galactosid-ase than the reU mutant during amino acidlimitation, I constructed, in the identical geneticbackground, a series of relA+/relA pairs, eachcarrying a lac operon with a particular promotermutation. The strains used and details of thegenetic construction of Q4 through Q15 are pre-sented in Table 1. The lac promoter mutationsPrla, Pr9, Prl2, and Pr13 (1) are derived in onestep from lacP+, whereas PrL8uv5 is a morecomplex alteration; the latter mutant, PrL8uv5,is a revertant of the L8 promoter mutation (19)containing three base pair changes in the lacpromoter region (15) and able to express lacenzymes at a very high level even when cellularcAMP levels are extremely low (29).

After valine addition and lac induction in theQ strains carrying lac promoter mutations, in nocase does the reU + strain make 70-fold more,8-galactosidase than relA, as observed withlacP+ (Table 3). In each instance of lac tran-scription initiation at a mutant promoter, thereLA +/relA ratio was reduced; the reduction wasmost dramatic with the promoter mutantPrL8uv5, for which the relA + strain producedonly five times as much ,B-galactosidase as therelA strain (Fig. 1B and Table 3). It must bebome in mind that all of the fl-galactosidasesynthesis measurements shown in the centercolumns of Table 3 were made on cultures towhich valine had been added before the addition

VOL. 145, 1981

412 PRIMAKOFF

C/) 15 / 105 -

z Q2 reaArelAocPrA4reIA IocprL8uv5

Li

140°10 TO6

0

5IG.5re1AdocPlL8uv5

03 reiAI;P+

0 20 40 60 0 20 40 60

MINUTES

FIG. 1. Kinetics of 1)-galactosidase synthesis fr-om (A) lacP+ and (B) lacPrL8uv5 during amino acid

limitation after valine addition and lac induction. Experimental protocol is described in footnote a, Table 2,and footnote a, Table 3.

of lac inducer. ppGpp levels were much higherin the relA + strain than in the relA strain duringthese amino acid-limiting conditions that pre-ceded and continued throughout the period oflac induction. In steady-state growth, on theother hand, there is a basal level ofppGpp whichis about the same in reUA+ and relA strains (4,20, 33). The data on the right side of Table 3show that the rates of,-galactosidase synthesisin steady-state growth were the same in reUAand reLA strains; this was true for lacPrla,lacPr9, lacPrl2, lacPrl3, and lacPrL8uv5.To determine whether these results on fl-ga-

lactosidase production were unique to the Qstrains or to the condition of isoleucine limita-tion, I constructed different reUl/reU pairs:UL120, UL121, UL128, and UL129 (Tables 1and 4). They carry a valS(Ts) mutation and thetwo promoters, lacP+ and P'L8uv5, which gavethe most extreme (high and low) relAU/relAratios in the Q-strain background. It is posibleto grow these strains in any of a variety of mediaat a permissive temperature (300C) and thenshift the cultures to a semirestrictive tempera-ture at which ppGpp will accumulate in thereU+ strain because of valyl-tRNA limitation.Table 4 shows the rate of ,B-galactosidasesynthesis per 60 min by the valS(Ts) strains atan appropriate semirestrictive temperature(37.8°C). The units of 18-galactosidase synthe-sized in the valS(Ts) strains can be comparedwith the units synthesized in the Q strains (Fig.1). In the valS(Ts) background, the relA strain

TABLE 2. cAMP levels during steady-state growthand amino acid limitationa

Growth condition IntracellularcAMP (,.f)b

Steady stateQ2, Q3 glucose alone .. ..... 0.56 ± 0.22c

Amino acid limitationQ2 glucose plus valine ....... 0.39 ± 0.18dQ3 glucose plus valine ... 0.34 ± 0.16da Cultures of strains Q2 and Q3, grown to early log

phase in M-63-glucose plus required amino acids, weredivided into two aliquots, at 10 min before induction100 jig of valine per ml was added to one aliquot, andat time 0, 1 mM IPTG was added to all aliquots.Intracellular cAMP levels were determined by theerythrocyte membrane-binding assay (10, 17).

b Mean ± standard deviation.cTwelve individual cAMP determinations were

made in 3 separate experiments.d SiX individual cAMP determinations for Q2 and

10 individual cAMP determinations for Q3 were madeat different time points during the first hour aftervaline addition in three separate experiments.

again produced far more fB-galactosidase thanthe relA strain when the lac promoter was wildtype, and again, this relAU/relA difference wasmuch lower when transcription was from thelacPrL8uv5 promoter. For both P+ andPrL8uv5, the relA +/reU ratios were smaller inthe UL strain background (Table 4) than in thepreviously described Q strains (Table 3); sincethe reU alleles in the two backgrounds wereindependently isolated in two different labora-

J. BACTERIOL.

ROLE OF GENE reLA+ IN lac OPERON REGULATION 413

tories (Table 1), this could indicate that the relAmutation present in the UL strains is more leaky.

Effect of externally added cAMP. Endog-enous levels of cAMP regulate lac enzyme syn-thesis (9, 35), and the rates of ,B-galactosidaseproduction can be increased in many growth

TABLE 3. Synthesis of /B-galactosidase from variouslac promoters during amino acid limitation and

steady-state growthaIsogenic pair Amino acid limita- Steady-stateof strains tion growth

Differ- Differ-ential ential

(reLAU+ lac rate of Ratio rate of RatiorelA J promoterJ -galac- (reLAU / P-galac- (relA +

tosidase reUA) tosidase relA)synthe- synthe-

sis s5s

Q21 P+ 1.5 70 23 0.95Q3J 0.021 24

Q4} PrL8uv5 10.9 5 31 0.92

Q5 ~~~2.2 34

Q61 prla 3.5 17 41 1.12Q7J 0.2 37

Q81 Pr9 5.7 16 52 1.18Q9J 0.36 44

Q12} pr12 4.1 17 38 0.87

Q13J 0.24 44

Q14 Pr'13 7.8 26 47 0.81Q15J 0.29 58

a Strains were constructed as described in the text.As described in footnote a, Table 2, cultures of eachstrain were grown, and valine was added. After induc-tion, ,B-galactosidase was assayed in valine-treated cul-tures at 20, 40, and 60 min and in steady-state growthcultures at points between 10 and 25 min by followingo-nitrophenol production at 420 nm from o-nitro-phenyl-f8-D-galactoside. f8-galactosidase units were cal-culated as previously described (22): 1 unit = (1,000x A4s0 [absorbance at 420 nm]) + (time of assay inminutes x ml of culture assayed x Asso of culture atthe time of induction). Uninduced values have beensubtracted. Residual amino acid incorporation in va-

line-treated Q4 and Q5, measured by incorporation of[3H]phenylalanine, was linear for 1 h and in both Q4and Q5 equalled 14% of the rate in the correspondingsteady-state growth cultures. Differential rate of syn-

thesis = (units of ,B-galactosidase synthesized per 60min) (relative amount of [3H]phenylalanine incor-porated per 60 min). The relative amounts of [3H]-phenylalanine incorporated were 14 for valine-treatedcultures and 100 for steady-state growth cultures. Therates for valine-treated Q2, Q3, Q4, and Q5 are theaverage of three experiments and, for valine-treatedQ6, Q7, Q8, Q9, Q12, Q13, Q14, and Q15, the average

of two experiments.

TABLE 4. /-galactosidase synthesis duringaminoacyl-tRNA limitation in valS(Ts) strains

,B-Ga-lactosi-dase Ratio

Streina Relevant genotype units (relA +synthe- reUA)sized/60 minb

UL120 lacP+ valS(Ts) relA 19.5 17UL121 lacP' valS(Ts) relA 1.17

UL128 lacPrL8uv5 valS(Ts) 42.7 2.7relA+

UL129 lacPrL8uv5 valS(Ts) 15.7relA

a UL120, UL121, UL128, and UL129 were con-structed by the steps described in footnote a, Table 1,from rpsL derivatives of the isogenic relA /relA pairof NP290291 and NP290292 (Table 1). Cultures inearly log phase in LB (22) at 300C were shifted to37.8°C at time 0, and 10 min later, 1 mM IPTG wasadded. Aliquots were removed for f,-galactosidase as-say between 10 and 25 min after induction.

b /?-Galactosidase units were calculated as describedin footnote a of Table 3, by using Auo of the culture atthe time of the temperature shift as the culture den-sity. Uninduced values have been subtracted; valuesare the average of three experiments.

conditions by the addition of cAMP to the me-dium (1, 10). In the condition of amino acidlimitation utilized here, endogenous levels ofcAMP were very low (Table 2); a non-physiolog-ical situation can be created by adding highconcentrations ofextemal cAMP to the medium,and this may expose other aspects of lac operonexpression in the amino acid-limiting condition.Table 5 shows the results of adding increasinglevels of cAMP to reA+/reA strains (Q2, Q3)during amino acid limitation. Rates of f8-galac-tosidase synthesis from lacP+ were markedlystimulated in both reA+ and reA by cAMPaddition, and the increase for any externallyadded cAMP level is larger in the mutant relAbackground. At saturating levels, 25 to 40 mMcAMP, fi-galactosidase synthesis was stimulated10-fold in Q2 reU+ and up to 200-fold in Q3reA. Control experiments showed that the ad-dition of 25 mM external cAMP during aminoacid limitation did not alter the level of ppGppin either Q2 or Q3 and did not alter the residualrate of protein synthesis in the two strains (datanot shown). Also, one obtains the same experi-mental result when cAMP is present for manygenerations before amino acid limitation is im-posed or is introduced 10 min after valine addi-tion with the lac inducer, IPTG. Thus, the addedcAMP did not seem to be acting by changingppGpp levels or by altering the growth proper-

VOL. 145, 1981

414 PRIMAKOFF

TABLE 5. Effect of exogenous cAMP on f,-galactosidase synthesis during amino acid

limitationaQ2 lacP+ relA Q3 lacP+ relA

Exter- Differen- /3-Galac- PGlcnal tial rate tosidaseDfee-tsds

cAMP Of/8-ga- synthesis tial rate Of syteiExter) lactosi- rate in- 8-galacto-crease in sidase rt ndase syn- rM snhiscrease in

thesis rl)s+ynthesis reLA (fold)

0 1.4 0.0210.03 2.6 1.9 0.064 3.00.1 5.4 4.0 0.57 26.71.0 7.4 5.4 0.61 27.75.0 9.8 7.3 2.1 96.7

25 14.4 10.6 3.6 16740 14.2 10.5 5.0 233

a The experimental procedure is described in foot-note a, Table 3. The given concentration of cAMP isadded at time 0 with 1 mM IPTG. Differential rate ofsynthesis was as defined in footnote a, Table 3; therelative amount of [3H]phenylalanine incorporatedwas 14 for valine-treated cultures. Values for ,B-galac-tosidase synthesis are the average of three experi-ments.

ties or protein synthetic capacities oi the strainsduring the amino acid-limiting condition.

DISCUSSIONThe goal of the present study was to ask

whether in vivo data on rates of lac enzymesynthesis would be consistent with recent invitro data showing a promoter-specific ppGppstimulation of lac transcription (25). In the invivo experiments, rates of ,B-galactosidase syn-thesis were measured in relA + cells and isogenicrelA cells during single amino acid limitation inthe continuing presence of carbon source. Thisexperimental approach revealed that rates of ,B-galactosidase synthesis in strains with the wild-type lac operon were 20- to 70-fold-higher in therelA+ strain than in the relA mutant (Fig. la;Tables 3 and 4). Since ppGpp accumulates tohigh concentration in relA+ strains under theamino acid limitation conditions used here (4),the elevation of ,B-galactosidase synthesis inreU+ suggests that high ppGpp levels in vivocan stimulate expression of lac operon enzymes.A key result of the current experiments was thatmutation in the lac promoter altered the amountof stimulation obtained in the reA+ strain. Allof the lac promoter mutations tested as com-pared with lacP+ showed a lower relAU/reLAratio for ,8-galactosidase synthesis rates (Tables3 and 4). Since the promoter is the site of tran-scription initiation, these experiments suggestthat, during amino acid limitation in vivo,

J. BACTERIOL.

ppGpp can stimulate transcription initiationfrom lacP+ and these particular mutants in thelac promoter are relatively independent of theppGpp stimulation.

In other work, we have tested this idea in vitroby examining ppGpp stimulations of lac expres-sion by using one DNA template carrying lacP+and a second DNA template carrying one of themutant lac promoters, PrL8uv5. Use has beenmade of the coupled transcription-translationsystem prepared from Salmonella typhimurium(2) in which ppGpp stimulation ofthe his operonmirrors in vivo demonstrated relA +/relA differ-ences in his enzyme synthesis (30). In the Sal-monella in vitro system, saturating levels ofppGpp stimulate ,B-galactosidase synthesis fromthe lacP+ template 20-fold; ,8-galactosidase syn-thesis from the lacP'L8uv5 template is high inthe absence of ppGpp and can be boosted lessthan twofold by the presence of saturatingppGpp levels (25). Thus, under appropriate invitro conditions, ppGpp acts to stimulate tran-scription of lac genes; the effect is large, and thepromoter specificity is the same as observed herein vivo, so that correspondence of the in vivoand in vitro data on these points is good.This set of results with the lac operon in vivo

and in vitro is consistent with a number ofrecentreports that identify specific genes (trp, his, ilv,ara) for which ppGpp may be a positive regu-lator (13, 14, 23, 30, 32, 34). The evidence sug-gests that ppGpp can stimulate expression ofboth biosynthetic and catabolic enzymes; thus asimple nutritional shift, single amino acid limi-tation, can potentially have very broad conse-quences for the control of cellular gene activitythrough the positive and negative actions of thisone regulatory nucleotide.

Additional control features of the lac operon(and possibly other systems) during amino acidlimitation have been revealed by examining en-dogenous cAMP levels and the effects of exoge-nous cAMP. The availability of a sensitivemethod for determining cAMP concentrationsin E. coli (10, 17) has allowed the measurementof the levels of this nucleotide after valine addi-tion. cAMP levels were quite low and essentiallythe same, about 0.35 ,uM, after valine addition inboth Q2 relA+ and Q3 relA (Table 2). Thisshows that there was no cAMP contribution tothe relA+/rel4 differntial in lac expression. Asurprising finding was obtained, however, whenthis low internal cAMP level was changed to ahigh non-physiological level, presumably alsothe same in both Q2 reA + and Q3 relA, byadding external cAMP. During amino acid lim-itation, high extemal cAMP stimulated Q2relA + 8-galactosidase synthesis 10-fold, whereasit stimulated Q3 relA /-galactosidase 200-fold

ROLE OF GENE reUA IN lac OPERON REGULATION 415

(Table 5). Control experiments showed that thelevels ofppGpp and rates of protein synthesis inQ2 and Q3 were unaffected by the high externalcAMP, so it is unclear what mechanisms may beinvolved in these effects.One possibility is that although relA mutants

lack endogenous ppGpp, cAMP can replaceppGpp in stimulating lac expression in relAduring amino acid limitation. Freundlich (13)has described a similar observation on cAMPstimulation of ilv operon expression in relA dur-ing amino acid limitation. Alternatively, the highcAMP and high ppGpp in the reLAU strain mayinteract in vivo such that the stimulation by onelimits the potential stimulation by the other. Incontrast, in in vitro experiments, the cAMP andppGpp stimulations of lac expression are inde-pendent, and both are required for maximalexpression (25). This difference between the invivo and in vitro experiments remains to beresolved. The stimulations observed in vivo byexternally added cAMP might reflect an unex-plored role of still other lac regulatory mole-cules, e.g., the catabolite modulator factor ofUllman et al. (31). At the present time it is notpossible to specify which in vivo physiologicalcorrelates of amino acid limitation are relevantin determining the relA+ and relA response tocAMP in the medium.

Earlier investigators of stringent control phe-nomena have reported evidence indicating trans-lational errors in the production of,-galactosid-ase in relA cells during amino acid limitation.Hall and Gallant (16) and Fill et al. (11) utilizedconditions of amino acid limitation coupled witheither carbon source starvation or high externalcAMP in relA+ and relA strains. The 18-galac-tosidase synthesized under these conditions andassayed in crude extracts of the relA cellsshowed greater thermolability than the ,B-galac-tosidase synthesized and assayed in reUA+ cells.Foley et al. (12) have now measured, in experi-ments using 5 mM (external) cAMP, a 13-folddifference for ,8-galactosidase synthesis betweenreLAU and relA strains. They find that roughlya fourfold difference can be attributed to a tran-scriptional difference and that a threefold differ-ence is due to a translational defect in relA. It isreasonable to suppose that translational effectsmay be present in our results too; with transcrip-tion occurring from lacPrL8uv5, the relA +/relAdifference is only three- to fivefold. This suggeststhat relA translational errors could be respon-sible for some or all of a fivefold effect. Themaximal 70-fold reLA +/relA difference observedfor wild-type lacP+ can then be seen as a com-pound result of the ppGpp stimulation of tran-scription initiation in relAU (about a 20-foldeffect in the in vitro assay) and a reduced activity

of tkhe /i-galactosidase synthesized in relA (pos-sibly a three- to fourfold effect).

ACKNOWLEDGMENTSI thank Jon Beckwith, in whose laboratory this work was

done, for teaching me a large number of important things andgiving me steady support and advice. I am very grateful toWolf Epstein, who allowed me to come to his laboratory anduse the expertise developed there in amaying cAMP in E. coli.I am indebted to Paul Schedl and Stan Artz for many valuablediscuaions and to Bob Perlnan for critically reading themanuscript. The assistance of Ronnie MacGillivray in makingmedia is gratefully acknowledged.

This work was supported by a National Science Foundationgrant (BMS 74 21663) to Jon Beckwith and a Helen HayWhitney Fellowship to the author.

LITERATURE CIE1. Arditti, R., T. Grodzicker, and J. Beckwith. 1973.

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3. Beckwith, J. R., T. Grodzicker, and R. R. Arditti.1972. Evidence for two sites in the lac promoter region.J. Mol. Biol. 69:155-160.

4. Cashel, M. 1969. The control of ribonucleic acid synthesisin Escherichia coli. IV. Relevance of unusual phospho-rylated compounds from amino-acid starved stingentstrains. J. Biol. Chem. 244:3133-3141.

5. Cashel, ML, and J. Gallant. 1974. Cellular regulation ofguanosine tetraphosphate and guanosine pentaphos-phate, p. 734-745. In M. Nomura and P. Lengyel (ed.),Ribosomes. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.

6. deCrombrugghe, B., G. Chen, M. Gottesman, L. Pas-tan, H. E. Varmus, M. Emmer, and R. L. Perlman.1971. Regulation of lac mRNA synthesis in a solublecell-free system. Nature (London) New Biol. 230:37-40.

7. Dennis, P. O., and ML Nomura. 1975. Stringent controlof the banscriptional activities of ribosomal proteingenes in E. coli. Nature (London) 255:460-465.

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9. Emmer, KL, B. deCrombrugghe, I. Pastan, and R. LPerlman. 1970. Cyclic AMP receptor protein of E. coli:its role in the synthesis of inducible enzymes. Proc.Natl. Acad. Sci. U.SA. 66:480-487.

10. Epstein, W., L B. Rothman-Denes, and J. E. Hesse.1975. Adenosine 3':5'-cyclic monophosphate as mediatorof catabolite repression in Escherichia coli. Proc. Natl.Acad. Sci. U.S.A. 72:2300-2304.

11. Fiil, N., B. KL Willumsen, J. D. Friesen, and K. vonMeyenberg. 1977. Interactions of alleles of the relA,relC, and spoT genes in Escherichia coli: analysis ofthe interconversion of GTP, ppGpp, and pppGpp. Mol.Gen. Genet. 150:87-101.

12. Foley, D., P. Dennis, and J. Gallant. 1981. Mechanismof the rel defect in 18-galactosidase synthesis. J. Bacte-riol. 145:641-643.

13. Freundlich, M. 1977. Cyclic AMP can replace the relA-dependent requirement for derepression of acetohy-droxy acid synthase in E. coli K-12. Cell 12:1121-1126.

14. Furano, A. V., and F. P. Wittel. 1977. Effect of the relAgene on depression of amino acid biosynthetic enzymesin growing Escherichia coli depends on the pathwaybeing derepressed. J. Bacteriol. 132:352-355.

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15. Gilbert, W. 1976. Starting and stopping sequences for theRNA polymerase, p. 193-205. In R. Losick and M.Chamberlin (ed.), RNA polymerase. Cold Spring Har-bor Laboratory, Cold Spring Harbor, N.Y.

16. Hall, B., and J. Gallant. 1972. Defective translation inRC- cells. Nature (London) New Biol. 237:131-135.

17. Hesse, J. E., L B. Rothman-Denes, and W. Epstein.1975. A convenient erythrocyte membrane cyclic AMPbinding assay. Anal. Biochem. 68:202-208.

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