Alterations in the β Flap and β′ Dock Domains of the RNA Polymerase Abolish NusA-Mediated Feedback Regulation of the metY - nusA - infB Operon

The RimM protein inEscherichia coli is important for thein vivo maturation of 30S ribosomal subunits and a ΔrimM mutant grows poorly due to assembly and translational defects. These deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, RbfA, encoded by themetY-nusA-infB operon. Among these suppressors are mutations innusA that impair the NusA-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of themetY-nusA-infB operon. We describe here the isolation of two new mutations, one inrpoB and one inrpoC (encoding the β and β′ subunits of the RNA polymerase, respectively), that increase the synthesis of RbfA by preventing NusA from stimulating termination at the internal intrinsic transcriptional terminators of themetY-nusA-infB operon. TherpoB2063 mutation changed the isoleucine in position 905 of the β flap-tip helix to a serine, while therpoC2064 mutation duplicated positions 415 to 416 (valine-isoleucine) at the base of the β′ dock domain. These findings support previously publishedin vitro results, which have suggested that the β flap-tip helix and β′ dock domain at either side of the RNA exit tunnel mediate the binding to NusA during transcriptional pausing and termination.