PhoP-P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP-P activator sites within the coding region stimulate transcription in vitro.
The Bacillus subtilis pstS operon and phoA gene are members of the Pho regulon that is controlled by PhoR, a histidine kinase, and PhoP, a response regulator. Footprinting analysis showed that phosphorylated PhoP extended the PhoP protected region in pstS and phoA promoters, and also bound to a separate site within the coding region of each gene. Our previous in vivo studies have shown that, in contrast to other Pho regulon promoters that are not expressed in either phoP or phoR mutants, a low-level induction from the pstS promoter (25% of parent strain) can be detected in a phoR mutant. In this study, by using an in vitro transcription system, we demonstrate that (i) only phosphorylated PhoP is a transcriptional activator of the pstS operon and of the phoA gene; (ii) phosphorylated PhoP and RNA polymerase sigmaA holoenzyme are sufficient for in vitro transcription of the pstS promoter and the phoA promoter; (iii) the activation of the pstS promoter requires lower concentrations of phosphorylated PhoP than does the phoA promoter for transcription; and (iv) PhoP binding sites in both the pstS promoter core binding region and in the 5' coding region of the gene, which have been identified by footprinting analysis, are important for the transcription of the pstS promoter in vitro.
Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
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Qi
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Hulett
D000469:Alkaline Phosphatase; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D003067:Coenzymes; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D018085:Regulon; D012808:Sigma Factor / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003067:Coenzymes; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D033902:Periplasmic Binding Proteins; D028044:Phosphate-Binding Proteins; C090133:PstS protein, E coli; D012808:Sigma Factor; C061279:PhoR protein, Bacteria; C061064:PhoP protein, Bacteria; D018844:Cyclin-Dependent Kinases; C063505:RNA polymerase sigma A; D012321:DNA-Directed RNA Polymerases; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli