Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa.
In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI-mediated activation of trpPB and TrpI-mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly-migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild-type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein--protein and protein--DNA interactions is required for activation of trpPB by TrpI and InGP.
Gao
J
J
Department of Biology, University of Iowa, Iowa City 52242.
Gussin
G N
GN
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016172:DNA Fingerprinting; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005994:Glycerophosphates / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D015534:Trans-Activators; D014158:Transcription, Genetic; D014367:Tryptophan Synthase / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D005994:Glycerophosphates; D015534:Trans-Activators; C008117:indoleglycerol phosphate; D014367:Tryptophan Synthase