Conformational changes of Escherichia coli RNA polymerase sigma(70) factorinduced by binding to the core enzyme

Mutants of RNA polymerase sigma(70) subunit from Escherichia coli with uniq ue cysteine residues engineered into conserved region I (autoinhibition dom ain of (sigma(70)), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between region s 1 and 2 were prepared. The chemical reactivity of the cysteine at each po sition was determined for free sigma(70) and sigma(70) 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 polymeras e. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma(70), providing d irect physical evidence for modulation of sigma(70) conformation by the cor e enzyme. Binding of the core polymerase resulted in increased solvent expo sure of DNA binding domains of sigma(70) and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma( 70) were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma(70). Thus, the results obtained provided ph ysical evidence supporting a model in which core enzyme allosterically regu lates DNA binding activity of sigma(70) by "unmasking" its DNA binding doma ins.