DNA-binding domain mutants of sigma-N (sigmaN, sigma54) defective between closed and stable open promoter complex formation.
The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function. Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes. The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.
Oguiza
J A
JA
Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
Buck
M
M
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development; D029968:Escherichia coli Proteins; D016296:Mutagenesis; D010641:Phenotype; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D010649:Phenylalanine; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54; D000409:Alanine