Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.     

redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD

redD and actII-ORF4, regulatory genes required for synthesis of the antibiotics undecylprodigiosin and actinorhodin by Streptomyces coelicolor A3(2), were transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. Disruption of hrdD had no effect on antibiotic production, indicating that redD and actII-ORF4 are transcribed in vivo by at least one other RNA polymerase holoenzyme. These data provide the first experimental evidence that HrdD can function as a sigma factor.     

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.