Sequences in sigma(54) region I required for binding to early melted DNA and their involvement in sigma-DNA isomerisation.
The bacterial sigma(54) RNA polymerase functions in a transcription activation mechanism that fully relies upon nucleotide hydrolysis by an enhancer binding activator protein to stimulate open complex formation. Here, we describe results of DNA-binding assays used to probe the role of the sigma(54) amino terminal region I in activation. Of the 15 region I alanine substitution mutants assayed, several specifically failed to bind to a DNA structure representing an early conformation in DNA melting. The same mutants are defective in activated transcription and in forming an isomerised sigma-DNA complex on the early opened DNA. The mechanism of activation may therefore require tight binding of sigma(54) to particular early melted DNA structures. Where mutant sigma(54) binding to early melted DNA was detected, activator-dependent isomerisation generally occurred as efficiently as with the wild-type protein, suggesting that certain region I sequences are largely uninvolved in sigma isomerisation. DNA-binding, sigma isomerisation and transcription activation assays allow formulation of a functional map of region I.
Gallegos
M T
MT
Department of Biology, Imperial College of Science Technology, and Medicine, Imperial College Road, London, SW7 2AZ, UK. m.gallegos@ic.ac.uk
Buck
M
M
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics; D020029:Base Pairing / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006493:Heparin / Q000494:pharmacology; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D020035:Holoenzymes; D009692:Nucleic Acid Heteroduplexes; D012808:Sigma Factor; D015534:Trans-Activators; D006493:Heparin; D010088:Oxidoreductases; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54