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 chromosome that contains the lac operon in E. coli, ously unavailable for verifying the significance of ppGpp effects on gene 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 genes, 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.