Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1726-1730, April 1979 Biochemistry Positive control of lac operon expression in vitro by guanosine 5'-diphosphate 3'-diphosphate (coupled protein synthesis in vitro/transcription initiation/supercontrol systems/overlapping metabolic domains/stringent phenomenon) PAUL PRIMAKOFF* AND STANLEY W. ARTZtf *Department of Microbiology and Molecular Geneties, Harvard Medical School, Boston, Massachusetts 02115; and tDepartment of Bacteriology, University of California, Davis, California 95616 and Department of Biochemistry, University of California, Berkeley, California 94720 Communicated by Bruce N. Ames, February 2, 1979

ABSTRACT Maximal expression of the Escherichia coli positive regulatory response to ppGpp of 3-galactosidase syn- lactose operon in a coupled in vitro transcription-translation thesis in vitro and have shown that this response is greatly di- system from a Salmonella typhimurium reIA mutant was strongly dependent upon addition of guanosine 5'-diphosphate minished by altering the DNA sequence of the wild-type lac 3'-diphosphate (ppGpp). Without added ppGpp, at saturating promoter. These results are considered in terms of overlap of 3',5'-cyclic AMP (cAMP) concentrations, synthesis of ft-galac- the 3',5'-cyclic AMP (cAMP) (5) and ppGpp (2) metabolic tosidase (P-D-galactoside galactoh drolase, EC 3.2.1.23) was domains (6). reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was MATERIALS AND METHODS uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. Bacterial Strains. S. typhimurium strain TA705 (his- When coupled I-galactosidase synthesis was primed with a AOGDCBH2253 hisT1504 relAl, refs. 2, 7) was used to pre- template containing a well-characterized mutant lac promoter pare in vitro protein-synthesizing extracts. In addition to this (-JacPrLAUV5), the dependence on ppGpp was greatly reduced. strain's having the his deletion, all Salmonella strains lack the This result provides an important experimental control previ- region of the that contains the lac operon in E. coli, ously unavailable for verifying the significance of ppGpp effects on regulation in vitro; it indicates that activation of lacr+ including the structural gene (lad) coding for the lac repressor expression by ppGpp is specifically an effect of increased (8). The following E. coli lysogens were used as sources of transcription initiations. Furthermore, the large ppGpp stimu- template DNA: TA1933 (streptomycin resistant, his-6607 [080h lation of acP+ DNA enabled the level of expression of this immX cd857 susS7, 080h immA cd857 susS7 dhis +], refs. 9, 10); template to approach that of IacPrL8UV5 DNA, an observation CSH44 (tonA lac.A thi [480h immX cd857 St68, 480h immX expected from results in vivo but not obtained with other tran- cI857 St68 dlacP+], ref. 11); and, RV/80 (tonA lacA thi [080h scription-translation systems in vitro. The importance of these \ results is considered with respect to previous ideas on the immX cd857 susS7, 080h imm cI857 susS7 dlacPrL8UV5], physiological role of ppGpp as a supercontrol molecule in bac- ref. 12,. the gift of W. Gilbert). terial regulation. Conditions for Protein Synthesis In Vitro. Reaction mix- tures (0.05 ml) were as described (2, 13) with the following The "stringent phenomenon" (1) in bacteria encompasses a changes: template DNA containing the lac operon was at a final complex array of both positive and negative regulatory re- concentration of 50-60 jtg/ml, and syntheses were initiated by sponses influenced primarily by availability of amino acids in addition of S-30 protein without preincubation of reaction the environment. The unusual nucleotide guanosine 5'-di- mixtures. Concentrations of cAMP and ppGpp are indicated phosphate 3'-diphosphate (ppGpp) is thought to be the general for each experiment. Because S-30 extracts from strain TA705 signal molecule in a supercontrol system that senses an imbal- are devoid of his and lac operon enzymes, it is possible to follow ance or deficiency in amino acid supply and redirects the cell's de novo production of histidinol dehydrogenase and fl-galac- economy in response (2). tosidase activities coded for by the hisD and lacZ , re- Previous studies utilizing DNA-dependent in vitro protein- spectively. synthesizing systems from Escherichia coli (3) had suggested Assay of Histidinol Dehydrogenase [L-Histidinol: NAD+ the possibility of positive control of gene expression by ppGpp Oxidoreductase, EC 1.1.1.23). Assay conditions and determi- for the catabolic lac and ara operons (4). Stimulation by ppGpp nation of ['4C]histidine from [14C]histidinol have been described in each case was small (about 2-fold) and in vivo confirmation (14). was lacking, making it difficult to assign a definite physiological Assay of fl-Galactosidase (#-D-Galactoside Galactohy- significance to the weak in vitro effects. drolase, EC 3.2.1.23). Protein synthesis was terminated by In the present study, we were prompted to reinvestigate the addition of 0.35 ml of Z buffer (11) to each 0.05 ml reaction effect of ppGpp on lac operon expression in vitro by two fac- mixture and the tubes were equilibrated at 28"C for 20 min. tors: (i) evidence suggesting an important regulatory role of The f3-galactosidase assay was initiated with 0.1 ml of o-nitro- ppGpp on expression of the lac operon in vtvo during amino phenyl-3-D-galactoside at 4 mg/ml, incubation was continued acid starvation (unpublished) and (ii) availability of a DNA- at 280C, and the reaction was stopped by addition of 0.25 ml dependent in vitro protein synthesizing system from Salmo- of 1 M sodium carbonate after sufficient yellow color had de- nella typhimurlum capable of giving positive regulatory re- veloped. Incubation times varied from 10 min to 10 hr and the sponses to ppGpp much greater (2) than those previously re- ported (4). Utilizing this system, we have found a marked Abbreviations: ppGpp, guanosine 5'-diphosphate 3'-diphosphate; cAMP, 3',5'-cyclic adenosine monophosphate; CAP, cyclic AMP- The publication costs of this article were defrayed in part by page binding protein. charge payment. This article must therefore be hereby marked "ad- t To whom reprint requests should be addressed. Present address: vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Department of Bacteriology, University of California, Davis, CA this fact. 95616. 1726 Downloaded by guest on September 27, 2021 Biochemistry: Primakoff and Artz Proc. Natl. Acad. Sci. USA 76 (1979) 1727

assay was linear over this time range. A precipitate was removed curred at 0.2 mM ppGpp and the concentration giving half- by 5-min centrifugation in a Beckman Microfuge and o-ni- maximal stimulation was about 0.06 mM. trophenol in the supernatant was measured by its absorbance Thus, the magnitude of the response of lac operon expression at 420 nm with a Zeiss PMQII spectrophotometer. Activities to added ppGpp is even greater than that of his operon ex- are normalized to 1 hr of incubation. pression, which was previously the largest demonstrated positive Reagents. Bulk tRNA was isolated (15) from wild-type S. effect of ppGpp on in vitro bacterial gene expression (2). In typhimurium strain TA265. ppGpp was prepared and analyzed addition, there is a shift in the concentration dependence for essentially as described (16, 17). ppGpp between the two operons, with the lac operon requiring 3-4 times more ppGpp than the his operon for either optimal or half-maximal expression. RESULTS The activation by ppGpp of lac expression we observed is ppGpp strongly activates lac operon expression in vitro markedly greater than the 2-fold stimulation previously re- ported in other studies (4) that utilized E. coli S-30 extracts Using a coupled transcription-translation in vitro system prepared to containing S-30 extract from a S. relA mutant, according Zubay (3). It has been shown (2) that typhimurium is ineffective in of the we found a striking dependence on the presence of ppGpp of ppGpp relatively stimulating expression his operon when added to an S-30 from a S. typhimurium de novo /3-galactosidase synthesis (assayed as a measure of lac relA + strain, apparently owing to endogenous ppGpp pro- operon expression). Fig. 1 indicates that, in the absence of added duction during protein synthesis. We therefore considered the ppGpp, in vitro lac operon expression primed with a DNA trivial possibility that failure of other studies to find a large template containing the wild-type lac promoter (lacP + ) was effect of added ppGpp on lac expression in vitro resulted from only about 5% of the level obtained at 0.7 mM ppGpp (i.e., endogenous ppGpp production. Accordingly, an S-30 was ppGpp stimulated about 19-fold). Half-maximal stimulation prepared essentially by the Zubay procedure (3) from E. coli occurred at 0.20 mM ppGpp. In numerous different experi- strain MZ9 (18), which is a derivative of the strain 514 used by ments with three independent S-30 preparations the magnitude Yang et al. (4) to study ppGpp effects on lac expression. With of the response was 14- to 20-fold, the optimal or saturating the S-30 from strain MZ9, lac expression was stimulated about concentration of ppGpp varied from 0.5 to 0.8 mM, and the 2-fold by added ppGpp, and endogenous accumulation of concentration giving half-maximal stimulation was in the range ppGpp was undetectable throughout the course of the reaction 0.15-0.25 mM. The concentration of cAMP (0.05 mM) was (19) (J. Stephens and P. Primakoff, unpublished data). In ad- saturating in these experiments. For comparison is shown (Fig. dition, strain MZ9 is sensitive to growth inhibition by amino acid 1) a typical response to added ppGpp of expression of the S. analogs diagnostic for the relA mutation (2). We conclude, typhimurium histidine operon (2). A 12-fold stimulation oc- therefore, that endogenous ppGpp production cannot account for the disparity between our results and those of Yang et al. (4) and that E. coli strain 514 and its derivatives are probably relA mutants.

2.0 _ 0

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> I 1.6 -

00

0 CD

4r.Q

(0 1.2.--

0 0.2 0.4 0.6 0.8 1.0 ppGpp, mM FIG. 1. Effect of various concentrations of ppGpp on syntheses A& A A AA AA in vitro of j3-galactosidase (0) and histidinol dehydrogenase (0). 7 I I l I Experimental points are expressed relative to values (- 1.0) obtained 0 0.2 0.4 0.6 0.8 1.0 in the absence of added ppGpp. Actual values in the absence of added wGpp, mM ppGpp, which are averages of duplicate determinations, are: 0.13 FIG. 2. Effect of various concentrations of ppGpp at different A420/hr for f-galactosidase and 1250 cpm/hr for histidinol dehydro- cAMP levels: without added cAMP (A), and at 6 AM (-), and 60 ,uM genase. (0) added cAMP. Downloaded by guest on September 27, 2021 1728 Biochemistry: Primakoff and Artz Proc. Natl. Acad. Sci. USA 76 (1979) Independence of ppGpp and cAMP stimulatory effects on lac operon expression Fig. 2 shows the effects on f3-galactosidase synthesis of in- creasing ppGpp concentrations at different levels of cAMP. At limiting (6 ,gM) and saturating (60 ,qM) cAMP concentrations the stimulations by ppGpp were about 14- and 16-fold, re- spectively, In the absence of added cAMP, lacP + expression was essentially'undetectable, and it was difficult to determine the magnitude of the ppGpp effect under these conditions. The expected strong dependence of lacP + expression on added U) cAMP (Fig. 2, Table 1) indicates that read-through transcription 0 or incorrect transcription initiations, which have been reported to contribute high background levels of f-galactosidase syn- thesis in other studies (20), did not occur here. 0~~~~~~ The data indicate that ppGpp and cAMP each stimulate lacP + expression in an independent fashion. However, it is not possible to rule out sequential activation-i.e., some cAMP 0 being required before ppGpp may exert its effect. 0.05- ppGpp acts at the level of lac operon transcription co In vitro uncoupling experiments (2, 13, 21) provided evidence that the requirement for ppGpp in lac operon expression is exerted at the level of transcription (Fig. 3). That is, when A~~~A transcription and translation were dissociated, ppGpp stimu- 0~ lated lac operon expression only when added while transcrip- 0 5 10 15 20 25 30 tion occurred. Consistent with recent results (22), there was a Time of addition, min severe diminution in accumulation of translatable lac mRNA FIG. 3. Effect of ppGpp on uncoupled #3-galactosidase synthesis when transcription in vitro was uncoupled from translation, in vitro. Amino acids were omitted from initial reaction mixtures and apparently owing to premature termination of RNA poly- then added, along with rifamycin SV at 20 4g/ml to inhibit further merase activity on the DNA template. However, activation by transcription initiations (and with or without 0.5 mM ppGpp), at the times indicated (in 2 pl), and incubation was continued for a total'of ppGpp appeared to be independent of this "polarity" effect and 70 min. 0, ppGpp added at zero time; A, no ppGpp added; @, ppGpp the relative magnitudes of stimulation by ppGpp were very added at time indicated. (Inset) Coupled controls. Amino acids were similar (about 18-fold) whether or not transcription was ac- added at zero time without rifamycin and with or without ppGpp (in companied by translation (Fig. 3 and its inset). Mutant lac promoter alters response to ppGpp Table 1 shows the effects of ppGpp and cAMP on lac operon ulation by 0.05 mM cAMP, the laCprL8UV5 template was ex- expression in vitro when 3-galactosidase synthesis was primed pressed at high levels with or without added cAMP and the with DNA templates containing either the wild-type lac pro- stimulation by 0.05 mM cAMP was less than 2-fold (Table 1, moter (lacP + ) or a well-characterized (23, 24, 25) mutant lac lines 1 and 2). promoter (IaCPrL8UV5). With the lacPrL8UV.5 promoter the The novel result demonstrated in Table 1 is that expression lac genes are known to be transcribed in the absence of the of the lacPrL8UV5 mutant promoter is also relatively unaf- cAMP/cAMP-binding protein (CAP) complex both in vivo (23) fected by ppGpp addition. While 0.6 mM ppGpp activates lacP + expression an additional 20-fold in the presence of sat- and in vitro (24), and this result was confirmed here. Thus, urating cAMP, expression of lacPrL8UV5 DNA is nearly in- whereas lacP+ expression was virtually undetectable in the dependent of this ppGpp requirement (Table 1, lines 2 and 4). absence of added cAMP and there was at least a 50-fold stim- An additional, important observation is that combined activa- tion of 1acP + expression in vitro by ppGpp and cAMP enables the level of expression of this template to approach closely that Table 1. Effects of ppGpp and cAMP on coupled expression of of the lacPrL8UV5 template (Table 1, line 4). This is in contrast lacP+ and lacPrL8UV5 DNA templates to results of Eron and Block (24), who reported coupled ex- pression in vitro of lacPrL8UV5 DNA to be 50-100 times fl-Galactosidase, A420/hr than that of lacP + DNA. The of the dis- DNA IacPrL8UV5 DNA greater significance Additions lacP+ parity between their results and ours will be considered None <0.003 (<0.02) 3.43 (22.9) below. cAMP 0.15 (-1.0) 5.03 (33.5) ppGpp S0.003 ('0.02) 4.65 (31.0) DISCUSSION cAMP + ppGpp 2.94 (19.6) 5.60 (37.3) Findings reported in this paper include marked activation by Reaction mixtures contained, where indicated, 0.05 mM cAMP and ppGpp of lacP + expression and altered response of the lac- 0.6 mM ppGpp. Each value is the average oftriplicate determinations, PrL8UV5 mutant promoter. We place great importance on which varied by 10% or less. For ease of comparison, numbers in pa- characterization of the lacPrL8UV5 promoter as ppGpp-in- rentheses are expressed' relative to lacP+ expression at saturating dependent because this clearly demonstrates an altered in vitro cAMP without added ppGpp (-1.0). Activities of IacP+ and lac- effect of ppGpp resulting from mutation in a gene regulatory prL8UV5 DNA templates in the cell-free system were independently In the results in vitro are consistent with analyzed with respect to optimal cAMP, ppGpp, DNA, tRNA, and region. addition, magnesium acetate concentrations. Optimal concentrations of these studies in vivo that demonstrate that induction of f3-galactos- variables were not detectably different for the two templates. idase is greatly reduced in relA mutants (which are unable to Downloaded by guest on September 27, 2021 Biochemistry: Primakoff and Artz Proc. Natl. Acad. Sci. USA 76 (1979) 1729 increase ppGpp synthesis rapidly) undergoing amino acid Comparison with Previous Studies of Coupled lac Ex- starvation as compared to otherwise isogenic relA + strains. This pression In Vitro. Regulation of the Lac operon has been ex- relative reduction in expression is largely eliminated by the tensively studied with transcription-translation systems in vitro, lacPrL8UV5 mutant promoter (unpublished data). Thus, the and several studies from different laboratories have measured results in vitro and in vivo taken together indicate that ppGpp ppGpp effects (4, 32-34). These studies are consistent in that is a potent and physiologically relevant positive effector of lac ppGpp invariably stimulated fl-galactosidase synthesis; how- operon expression. In addition, activation in vitro of the cata- ever, the published data demonstrate only 2- to at most 5-fold bolic lac operon occurs at significantly higher levels of ppGpp effects. We feel that the failure of other studies to find the than that of the amino acid biosynthetic his operon (Fig. 1), striking activation by ppGpp we observe reproducibly for the supporting the concept of a hierarchy (2) of control among the lac, his, and trp (S. Jovanovich and S. Artz, unpublished results) systems regulated by this molecule. operons may involve loss of the postulated ppGpp activation Mechanism of ppGpp as a Positive Effector in Regulating factor(s) during preparation of S-30 extracts. Transcription Initiations at the lac Promoter. The lac- The work of Eron and Block (24) strongly supports the notion PrL8UV5 template contains three base-pair changes in the lac of a missing component. They reported lacPrL8UV5 to elicit regulatory region (25). One mutation (L8) is a single base-pair at least 50 times more fl-galactosidase synthesis than lacP+ with change in the CAP binding site (25-27). The other change a Zubay system (3) under conditions of saturating cAMP (UV5) alters two adjacent base pairs in the characteristic hep- (ppGpp effects were not studied) and designated lacPrL8UV5 tanucleotide sequence (28) just prior to the point of transcription a "super-promoter." Using an S-30 prepared according to initiation [5'-T-A-T-G-T-T-G-3' (lacP+) - 5'-T-A-T-A-A- Zubay from E. coLi strain MZ9, we have observed about a 15- T-G-3' (lacPrUV5)] (25). UV5 was isolated as a suppressor of fold difference in expression of the two templates even in the the L8 defect (23, 29) and enables RNA polymerase to initiate presence of ppGpp (unpublished results). This is in striking lac transcription in the absence of cAMP/CAP (23, 24). Because contrast to results shown in Table 1, in which lacP + expression the three base-pair changes in the the lacPrL8UV5 template approaches within a factor of two of that of laCPrL8UV5. In are all located before the transcription initiation site, effects of vivo, although resistant to "catabolite repression," the lac- ppGpp on lac mRNA elongation, degradation, or translation PrL8UV5 promoter is not a superpromoter. Depending on are eliminated, leaving only transcription initiation as the target growth conditions, maximally induced f3-galactosidase synthesis of ppGpp regulation. By converting the lacP+ heptanucleotide is no greater for lacPrL8UV5 than for lacP + (23), suggesting sequence to one closely resembling the so-called canonical that the superpromoter effect in vitro (24) is an artifact of the tight-binding sequence (5'-T-A-T-R-A-T-G-3') (R = purine) cell-free system. Our finding of increased ppGpp activation (28) the UV5 base-pair changes apparently generally obviate allowing lacP+ to approach lacPrL8UV5 expression, thus the requirement for positive control factors at the lac promoter. greatly reducing this superpromoter effect, is consistent with A plausible, tentative hypothesis is, therefore, that ppGpp the idea that a required ppGpp activation factor is missing from stimulates lacP + transcription initiations through interaction the cell-free systems used in other studies. The in vitro tran- with a regulatory activation factor or factors. scription-translation system we used was originally designed It seems unlikely that the postulated activation factor(s) is to investigate regulation of the S. typhimurium his operon (2, CAP or RNA polymerase itself because ppGpp does not appear 13). Obtaining active coupled synthesis with S-30s from S. ty- to stimulate lac mRNA synthesis in a minimal transcription phimurium strains required significant modification of the system containing only these proteins (30). Arguments for a preparative procedures commonly in use (3, 35), and this will ppGpp activator protein (GAP) binding to DNA are tempered be reported in detail elsewhere. In general, several of the somewhat by the observation that there is probably not room changes would be expected to protect unstable components in in the DNA sequence of the Lac regulatory region (25, 31) to the system (e.g., use of a protease inhibitor). contain an independent binding site in addition to those for Physiological Role of ppGpp in Regulating Expression of CAP, RNA polymerase, and lac repressor. However, overlap- Catabolic Genes. It has been known for many years that amino ping sites would be possible, and it is interesting to point out that acid starvation-which is the physiological signal for the there is available a sequence displaying twofold rotational stringent response (reviewed in ref. 1)-also leads to catabolite symmetry overlapping with the CAP binding site (25). When repression (reviewed in ref. 36) of genes involved in carbon DNA sequences of other ppGpp-activated promoters (e.g., the source utilization [the E. coLi lac operon has been most studied his operon promoter) are known, it will be important to analyze (37)]. In the case of transient or permanent catabolite repression them for similarities with this lac sequence. resulting from quality and quantity of carbon sources in the Aboud and Pastan (30) have reported partial purification of environment, regulatory responses are directly related to in- a transcription factor indirectly able to stimulate lac mRNA tracellular cAMP levels (38). Although we are unaware of direct synthesis in vitro, apparently by relieving the inhibition caused measurements of cAMP levels during amino acid starvation, by adding tRNA to their transcription system; addition of we think it probable that repression of catabolic genes as a ppGpp further enhanced the stimulation. Whether this factor consequence of amino acid starvation also results largely from is somehow related to our results is unclear, but it seems unlikely a decline in the intracellular concentration of cAMP. This at this time. First, the magnitude of ppGpp stimulation ob- concept is supported by evidence indicating that addition of served by Aboud and Pastan was much smaller than we found, cAMP to the growth medium (an experimentally useful, but and, second, their factor stimulated transcription of phage genes probably nonphysiological condition) during amino acid star- and lac genes to similar extents, whereas we find activation by vation greatly relieves the repression of lac operon expression ppGpp to be selective for bacterial gene expression (ref. 2; (ref. 39; unpublished data). unpublished results). Repression of the lac operon in vivo during amino acid Our working model is that the postulated ppGpp activation starvation is much stronger in reLA mutants than in reLA + factor is a protein that interacts either with DNA or with RNA strains (unpublished data). Correspondingly, our in vitro results polymerase. More complicated mechanisms, however, are demonstrate that ppGpp is capable of eliciting extensive acti- certainly possible; isolation of the required component(s) should vation of lac transcription initiations. We therefore propose that be enlightening in this respect. reLA +-dependent elevation of intracellular ppGpp levels during Downloaded by guest on September 27, 2021 1730 Biochemistry: Primakoff and Artz Proc. Natl. Acad. Scs'. USA 76 (1979) amino acid limitation can allow partial escape of lac operon 5. Alper, M. & Ames, B. N. (1978) J. Bacteriol. 133, 149-157. expression from the repressing effects of a concomitant decline 6. Tomkins, G. M. (1975) Science 189,760-763. in cAMP levels. It seems likely that this compensatory function 7. Martin, R. G. (1968) J. Mol. Biol. 31, 127-134. of ppGpp will apply to regulation of other catabolic genes and 8. Yagil, E. & Hermoni, E. (1976) J. Bacteriol. 128, 661-664. operons as well-e.g., the ara operon (4). 9. Smith, G. R. & Tong, B. (1974) J. Bacteriol. 120, 1223-1226. The role ascribed to ppGpp in activating expression of certain 10. Voll, M. J. (1972) J. Bacteriol. 109, 741-750. catabolic genes during amino acid starvation is related to pre- 11. Miller, J. H. (1972) Experiments in Molecular (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). vious ideas on events occurring during the stringent response 12. Maizels, N. M. (1973) Proc. Nati. Acad. Sci. USA 70, 3585- in bacteria. It has been argued (2) that elevated ppGpp levels 3589. resulting from physiological amino acid starvation (e.g., amino 13. Artz, S. W. & Broach, J. R. (1975) Proc. Natl. Acad. Sci. USA 72, acid downshift) signal the cell to turn on amino acid-producing 3453-3457. processes such as protein breakdown (40) and amino acid bio- 14. Ciesla, Z., Salvatore, F., Broach, J. R., Artz, S. W. & Ames, B. N. synthesis (2). At the same time ppGpp acts directly or indirectly (1975) Anal. Biochem. 63,44-55. to inhibit numerous high-energy-requiring processes such as 15. Brenner, M. & Ames, B. N. (1972) J. Biol. Chem. 247, 1080- ribosome (41), cell membrane (42), and cell wall (43) macro- 1088. molecular syntheses, which are otherwise in excess of need 16. Cashel, M. (1974) Anal. Biochem. 57, 100-107. during the period of growth-rate limitation due to decreased 17. Cashel, M. (1969) J. Biol. Chem. 244,3133-3141. 18. Inouye, H., Pratt, C., Beckwith, J. & Torriani, A. (1977) J. Mol. amino acid supply. These inhibitory responses enable the cell Biol. 110, 75-87. to channel resources into amino acid production and more ef- 19. Haseltine, W. A., Block, R., Gilbert, W. & Weber, K. (1972) ficiently overcome the deficit. Likewise, it can be suggested that Nature (London) 238,381-384. the apparent decline of intracellular cAMP concentration 20. Zubay, G., Chambers, D. A. & Cheong, L. C. (1970) in The during amino acid starvation would serve to diminish unnec- Lactose Operon, eds. Zipser, D. & Beckwith, J. R. (Cold Spring essary processes normally activated by this molecule. The ac- Harbor Laboratory, Cold Spring Harbor, NY), pp. 375-391. tivity of some cAMP-controlled processes, however, must be 21. Schumacher, G. & Ehring, R. (1973) Mol. Gen. Genet. 124, preferentially maintained for continued amino acid produc- 329-344. tion-e.g., expression of the lac operon if lactose is the carbon 22. Jacobs, K. A., Shen, V. & Schlessinger, D. (1978) Proc. Natl. Acad. source-and in such cases ppGpp would substitute for Sci. USA 75, 158-161. 23. Silverstone, A. E., Arditti, R. R. & Magasanik, B. (1970) Proc. cAMP. Natl. Acad. Sci. USA 66,773-779. Although our rationale for a regulatory design is speculative, 24. Eron, L. & Block, R. (1971) Proc. Natl. Acad. Sci. USA 68, the evidence does clearly indicate interaction between the 1828-1832. ppGpp (amino acid production) and cAMP (carbon and energy 25. Gilbert, W. (1976) in RNA Polymerase, eds. Losick, R. & source utilization) supercontrol systems. Similar reasoning has Chamberlin, M. (Cold Spring Harbor Laboratory, Cold Spring been applied to account for escape from catabolite repression Harbor, NY), pp. 193-205. of genes involved in nitrogen-source utilization during nitrogen 26. Majors, J. (1975) Nature (London) 256,672-674. starvation (44). In the case of general nitrogen control, however, 27. Arditti, R. R., Scaife, J. & Beckwith, J. (1968) J. Mol. Biol. 38, a molecule analogous to ppGpp or cAMP has not yet been dis- 421-426. covered. While such supercontrol or 28. Pribnow, D. (1975) Proc. Natl. Acad. Sci. USA 72,784-788. molecules, "symbols" (6), 29. Beckwith, J., Grodzicker, T. & Arditti, R. (1972) J. Mol. Biol. 65, "alarmones" (2, 5), are involved primarily in regulation of 155-160. defined metabolic domains (6), it appears that the domains also 30. Aboud, M. & Pastan, I. (1975) J. Biol. Chem. 250,2189-2195. overlap, providing an additional level of complexity in the 31. Dickson, R., Abelson, J., Barnes, W. & Reznikoff, W. (1975) analysis of regulatory networks. Science 187, 27-35. We thank Bruce Ames, in whose laboratory this in vitro work was 32. de Crombrugghe, B., Chen, B., Gottesman, M. & Pastan, I. (1971) initiated, and for their encouragement and support. Nature (London) New Biol. 230,37-40. S.W.A. is grateful to Bruce Ames for introducing him to many of the 33. Kung, H.-F., Fox, J. E., Spears, C., Brot, N. & Weissbach, H. ideas concerning how "alarmones" may be generally involved in (1973) J. Biol. Chem. 248,5012-5015. bacterial regulation. P.P. was a postdoctoral fellow in Jon Beckwith's 34. Kung, H.-F., Brot, N., Spears, C., Chen, B. & Weissbach, H. laboratory during the course of this work and was a Fellow of the (1974) Arch. Biochem. Biophys. 160, 168-174. Helen Hay Whitney Foundation. This research was supported by 35. Nirenberg, M. W. (1963) Methods Enzymol. 6, 17-23. National Institutes of Health Grant GM19993 to Bruce Ames and by 36. Pastan, I. & Adhya, S. (1976) Bacteriol. Rev. 40,527-551. the following support to S.W.A.: Special Grant 888 from the California 37. Nakada, D. & Magasanik, B. (1964) J. Mol. Biol. 8, 105-127. Division of the American Cancer Society, a grant from the Cancer 38. Epstein, W., Rothman-Denes, L. B. & Hesse, J. (1975) Proc. Natl. Research Coordinating Committee (University of California), and a Acad. Sci. USA 72,2300-2304. gift from Eli Lilly. 39. Kennell, D. & Simmons, C. (1972) J. Mol. Biol. 70,451-464. 40. Sussman, A. J. & Gilvarg, C. (1969) J. Biol. Chem. 244, 6304- 1. Gallant, J. & Lazzarini, R. A. 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