Kinetic properties of rrn promoters in Escherichia coli.
How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter-lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [R(f)], and the ratio of the Michaelis-Menten parameters, V(max)/K(m) (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD(460)) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD(460) and corresponds to an intracellular concentration of about 50 microM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.
Zhang
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Department of Molecular and Cell Biology, University of Texas at Dallas, TX 75083-0688, Richardson, USA.
Dennis
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Ehrenberg
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Bremer
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D004275:DNA, Ribosomal / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D035021:Factor For Inversion Stimulation Protein / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006159:Guanosine Tetraphosphate / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
D004275:DNA, Ribosomal; D035021:Factor For Inversion Stimulation Protein; D012335:RNA, Ribosomal; D006159:Guanosine Tetraphosphate; D012321:DNA-Directed RNA Polymerases