The sigma 54 DNA-binding domain includes a determinant of enhancer responsiveness.
The bacterial sigma54 protein associates with core RNA polymerase to form a holoenzyme that functions in enhancer-dependent transcription. Isomerization of the sigma54 polymerase and its engagement with melted DNA in open promoter complexes requires nucleotide hydrolysis by an enhancer-binding activator. We show that a single amino acid substitution, RA336, in the Klebsiella pneumoniae sigma54 C-terminal DNA-binding domain allows the holoenzyme to isomerize, engage with stably melted DNA and to transcribe from transiently melting DNA without an activator. Activator responsiveness for the formation of stable open complexes remained intact. The activator-independent transcription phenotype of RA336 is shared with mutants in amino-terminal Region I sequences. Thus, in sigma54, two distinct domains function for enhancer responsiveness. A sigma54-DNA contact mediated by R336 appears to be part of a network of interactions necessary for maintaining the transcriptionally inactive state of the holoenzyme. We suggest activator functions to change these interactions and facilitate open complex formation through promoting polymerase isomerization.
Chaney
M
M
Department of Biology, Biomedical Sciences Building, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK.
Buck
M
M
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004789:Enzyme Activation; D020868:Gene Silencing; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D012808:Sigma Factor; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54