Conformational changes of Escherichia coli RNA polymerase sigma70 factor induced by binding to the core enzyme.
Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70. Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains.
Callaci
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104, USA.
Heyduk
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Heyduk
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D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine