Proc. Nat. Aced. Sci. USA Vol. 72, No. 11, pp. 4389-4393, November 1975 Biochemistry Guanosine 5'-diphosphate 3'-diphosphate (ppGpp): Positive effector for histidine operon transcription andgeneral signal for amino-acid deficiency (relaxed-stringent control/alarmones/Salmonella typhimurium/super-control/coupled in vitro protein synthesis) JOHN C. STEPHENS, STANLEY W. ARTZ, AND BRUCE N. AMES Department of Biochemistry, University of California, Berkeley, Calif. 94720 Contributed by Bruce N. Ames, September 5, 1975 ABSTRACT Maximal expression of the histidine operon pression experiments; and TA1995 (dhuAl) and TA1996 of Salmonella typhimurium in a coupled in vitro transcrip- (dhuAl relAl)-amino-acid analog studies. The rel-1 muta- tion-translation system is strongly dependent upon addition tion (8) has been designated a relA mutation (9) on the basis of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). This re- quirement for ppGpp is exerted at the level of transcription of genetic mapping, phenotype, and biochemical character- through a mechanism distinct from the his-operon-specific ization (unpublished experiments). The dhuAl mutation regulatory mechanism. In vivo derepression of the his oper- (10) was used to facilitate strain construction and is not per- on is markedly defective when histidine starvation is im- tinent to these studies. The following Escherichia coli lyso- posed on a relA mutant-unable to rapidly increase synthesis gens (11) were used as source of template DNA: TA1933 of ppGpp-growing in amino-acid-rich medium. Increased [his-6607 StrR h immX cI857 susS7, (80 h dhisO+ sensitivity of relA mutants to growth inhibition by a number (080 of amino-acid analogs suggests that pp-pp is generally im- immA cI857 susS7)]; and TA1940 [his-6607 StrR ()80 h portant in adjusting expression of amino-acid-producing sys- immX cI857 susS7, 4i80 h dhisO1242 immx cI857 susS7)]. tems. Analysis of these findings leads us to propose that The intact his operon contained in each of the transducing ppcpp is a positive effector in a system that enables the cell phages originated from S. typhimurium genetic material to balance endogenous amino-acid production with environ- (12). mental conditions of amino-acid availability, and to compen- Conditions for In Vitro Protein Synthesis. Except where sate efficiently for transient changes in these conditions. We propose a unifying theory of the role of ppGpp as the general indicated, 50 gl reaction mixtures contained: Tris-acetate, signal molecule (alarmone) in a "super-contro " which senses 53 mM (pH 8.0); potassium acetate, 58 mM; ammonium ac- an amino-acid deficiency and redirects the cell's economy in etate, 27 mM; magnesium acetate, 10 mM; calcium acetate, response. 5 mM; dithiothreitol, 1.3 mM; 20 amino acids, 0.2 mM each; bulk tRNA, 0.5 mg/ml; ATP, 2 mM; CTP, GTP, UTP, 0.5 The "stringent phenomenon" in bacteria (1) has been pri- mM each; phosphoenolpyruvate, 20 mM; folinic acid, 30 marily interpreted as being a regulatory mechanism for ad- jig/ml; polyethylene glycol 6000, 35 mg/ml; p-toluenesulfo- justing the rate of synthesis of stable RNA (i.e., rRNA and nyl fluoride, 30 ,ug/ml; template DNA, 150 ,ug/ml; S-30 pro- tRNA) with respect to the availability of amino acids for tein, 3.5 mg/ml. Reaction mixtures, minus S-30 protein, protein synthesis. Evidence has accumulated suggesting that were incubated 3 min at 370 before the protein was added the unusual nucleotide guanosine 5'-diphosphate 3'-diphos- to initiate synthesis. Incubation at 370 was continued for 70 phate (ppGpp)-synthesized by the relA gene product on min and stopped by addition to the histidinol dehydrogenase the ribosome as a function of tRNA charging (2)-acts as a assay mixture. negative effector in this regulation, although the precise Concentrations given for magnesium acetate, calcium ac- mechanism is unclear. Comparison of rel+ and relA bacteri- etate, tRNA, and S-30 protein are only approximate and are al strains has revealed that ppGpp also may negatively con- determined for each protein synthesizing extract prepared. trol other aspects of cellular metabolism: biosynthesis of lip- Analysis of optimal conditions for in uitro protein synthesis, ids (3), nucleotides (4), and polyamines (5); and uptake of and methods for preparing Salmonella protein-synthesizing purines and pyrimidines (6, 7). extracts and template DNA, are contained in a separate re- In this paper we show that ppGpp is also a positive effec- port*. tor for transcription of the histidine operon of Salmonella Assay Methods and Reagents were: histidinol dehydro- typhimurium, and that it apparently positively regulates genase (L-histidinol:NAD oxidoreductase, EC 1.1.1.23) ref. production of other amino acids. 13; histidinol phosphate phosphatase (L-histidinolphosphate phosphohydrolase, EC 3.1.3.15) ref. 14; 6-phosphogluconate MATERIALS AND METHODS dehydrogenase (6-phospho-D-gluconate:NADP oxidoreduc- Bacterial Strains. Isogenic pairs of S. typhimurium tase, EC 1.1.1.43) ref. 15; and ppGpp ref. 16. Total in vitro strains were employed for the purpose indicated: TA471 protein synthesis was measured as incorporation of a mixture (his AOGDCBH2253 hisT1504) and TA705 (his- of 13 '4C-labeled amino acids (Schwarz Bioresearch) into AOGDCBH2253 hisT1504 relAl)-preparation of in vitro hot-acid-insoluble material*. Bulk tRNA was isolated (17) protein synthesizing extracts; TA2383 (hisG46 dhuAl) and from S. typhimurium strain TA253 (hisT1504). ppGpp was TA2384 (hisG46 dhuAl relAl)-in vivo his operon dere- prepared by Cashel's procedure (18). Abbreviations: ppGpp, guanosine 5'-diphosphate T3'-diphosphate; pppGpp, guanosine 5'-triphosphate 3'-diphosphate. * S. W. Artz, J. R. Broach, and B. N. Ames, in preparation. 4389 Downloaded by guest on September 25, 2021 4390 Biochemistry: Stephens et al. Proc. Nat. Acad. Sci. USA 72 (1975) 0 E E 0 01 o E E 0IE I -J i2.0 I I4 0~~~~~ 0a H 0 -i 0 0 0.1 02 03 04 0.5 *ppGpp (mM) 0 5 10 15 20 25 30 Time of Addition (min) FIG. 1. Effect of ppGpp on in vitro histidinol dehydrogenase FIG. 2. Effect of ppGpp on uncoupled in vitro protein synthe- (HDH) synthesis (0, q80dhis01242 DNA; 0, DNA) 080dhisO+ sis with S-30 from TA705 and and on total in vitro protein synthesis (A, 480 DNA). The S-30 ex- (relA) k80dhis01242 DNA. Amino acids were tract was from TA705 (relA). deleted from initial reaction mixtures and then added, along with 20 Mg/ml of rifamycin SV to inhibit further transcrip- RESULTS tion initiations (and with or without 0.2 mM ppGpp) at the times indicated (in 2 ,ul), and incubation was continued for a total of 70 ppGpp is required for maximal in vitro his operon min. Symbols: 0, ppGpp added at zero time; ,, no ppGpp added; expression *, ppGpp added at time indicated. Inset: Coupled control. Amino acids were added at zero time without rifamycin (and with or with- We utilize an in vitro coupled protein synthesizing system out ppGpp). which contains an S-30 cell extract, his operon DNA, and other components necessary for transcription and transla- tion. De novo synthesis of histidinol dehydrogenase [product stimulation with relA extracts, even though independently of the second structural gene (hisD) of the operon] serves as prepared S-30 extracts may vary considerably in protein- a measure of coupled his operon expression. Fig. 1 shows synthesizing activity. The lesser dependence of his operon that his operon expression is strongly dependent upon addi- expression on added ppGpp with S-30 extracts from the rel+ tion of ppGpp when S-30 extract from a relA mutant is used: strain is accounted for by the ability of such extracts to pro- a stimulation of about 10-fold is observed. Functional speci- duce and accumulate ppGpp during coupled protein synthe- ficity for this stimulation is indicated by lack of stimulation sis while S-30 extracts from the relA mutant fail to do so by ppGpp of total protein synthesis when phage 080 DNA is (Table 1). used as template (Fig. 1). In addition, other guanine nucleo- Requirement for ppGpp is independent of his operon- tides tested (GDP, cyclic GMP, or excess GTP-each at 0.1 specific controlling elements mM) are without significant effect. Fig. 1 demonstrates that ppGpp stimulates in vitro expres- In contrast, when S-30 extract from an isogenic rel+ strain is used, the requirement for ppGpp to obtain maximal sion of hisO1242 DNA about as much as wild-type (hisO+) his DNA. in vitro his operon expression is diminished (less than 2-fold operon The hisO1242 mutation deletes the "atten- stimulation) (Table 1). This lesser ppGpp uator" site in the hisO region of the his operon and results in stimulation by loss of the control mechanism with rel+ extracts is consistently observed, as is the marked complete operon-specific (19). We conclude, therefore, that the ppGpp requirement is ex- Table 1. Synthesis of ppGpp and pppGpp erted at a site other than the attenuator site, and that the during coupled in vitro protein synthesis regulatory function of this molecule is not part of the his- operon-specific control mechanism. This situation parallels ppGpp pppGpp HDH that observed for catabolite-sensitive operons, in that cAMP S-30 Conditions* nmol/ml pmol/ml regulation is effected through a mechanism distinct from rel+ Complete 18 22 1.04 operon-specific regulatory mechanisms (20). rel+ - Amino acids 30 37 <0.01 ppGpp acts at the level of his operon transcription rel+ + 0.2 mM ppGpp 1.52 relA Complete 3.5 1.5 0.70 In vitro uncoupling experiments (19, 21) provide evidence relA - Amino acids 3.0 1.2 <0.01 that the requirement for ppGpp in his operon expression is relA + 0.2 mM ppGpp 6.90 exerted at the level of transcription (Fig.