Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

Abstract

We have isolated mutations in rpoA, the gene encoding the a subunit of RNA polymerase, that specifically affect transcriptional control by OmpR and EnvZ, the two-component regulatory system that controls porin gene expression in Escherichia coli. Characterization of these mutations and a previously isolated rpoA allele suggests that both positive and negative regulation of porin gene transcription involves a direct interaction between OmpR and RNA polymerase through the a subunit. Several of the rpoA mutations cluster in the carboxy-terminal portion of the a protein, further suggesting that it is this domain of a that is involved in interaction with OmpR and perhaps other transcriptional regulators as well.Several different mechanisms have been proposed to explain how regulatory proteins function to catalyze transcription initiation (1). A common feature of many of these models is a direct physical interaction between the activator protein and the transcription apparatus. Evidence supporting such an interaction has been presented for several prokaryotic regulatory proteins. CRP, the cyclic AMP (cAMP) receptor protein, is an activator of many genes and operons and controls expression of lac, for example, by facilitating the binding of RNA polymerase (23). cI, A repressor, regulates its own expression by enhancing open complex formation at PRM (17,34). In this case as well, binding studies suggest a direct interaction (18). NR1 (NtrC) is the effector of the two-component regulatory system that stimulates expression of the genes of the nitrogen regulon. It activates the glnAp2 promoter, for example, via a mechanism that involves DNA looping (40). A complete understanding of transcriptional regulation requires a more detailed description of these activator-RNA polymerase interactions. In the case of cI, genetic studies suggest a region of the repressor molecule that is involved in contacting RNA polymerase(6). However, in all cases, little is known about the oppos-ing contact region of the polymerase. We have sought to address this question in our studies of the two-component system that regulates porin gene expression in Escherichia coli.The two-component regulatory system, OmpR and EnvZ, controls the differential expression of the porin genes, ompF and ompC, in response to medium osmolarity. EnvZ is a receptor kinase that functions as a sensor of osmolarity and communicates this information to the effector, OmpR (an NR1 homolog), by a mechanism involving phosphorylation and dephosphorylation. OmpR-P controls transcription initiation by binding to sites upstream of the appropriate promoters (19). We have shown previously that OmpR works in a positive fashion at the ompC promoter and both positively and negatively at the ompF promoter (37, 38). In media of low osmolarity, levels of OmpR-P are low and * Corresponding author. ompF expression is activated. In media of high osmolarity, levels of OmpR-P increase, ompC expression is activated, and ompF expression is repressed (11). Mutations in envZ that result in increas...

Continuous Control in Bacterial Regulatory Circuits

“…Promoter swap (30) and OmpF overexpression (39) experiments were consistent, at least qualitatively, with the absence of feedback. In addition, transcriptional reporter strains in which either ompC or ompF was disrupted with an insertion of lacZ showed osmoregulation of beta-galactosidase activity (44). However, the conclusions from other studies of ompF deletions were less clear (35,39).…”

Mechanism of gene activation by the Escherichia coli positive regulator, OmpR: A mutant defective in transcriptional activation

“…It has been reported that certain amino acid substitutions in the ~ subunit of RNA polymerase severely affect OmpRdependent activation of in vivo ompC and/or ompF transcription [5][6][7], A reconstituted RNA polymerase containing C-terminally truncated ~ subunits does not respond to in vitro transcription activation by certain activator proteins including OmpR [4]. These two independent lines of evidence strongly suggested that OmpR interacts with RNA polymerase (e.g.…”

“…the phosphorylation of OmpR results in remarkable enhancement of its DNA-binding ability [3]. Several lines of evidence suggested that OmpR functions through a direct interaction with RNA polymerase [4][5][6][7]. A crucial question then arose, namely, whether or not there is any contact site (or activation site) with RNA polymerase in the primary sequence of the OmpR molecule.…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…In addition to the RNAPH s 4 domain, the interactions between the C-terminal domain of the a-subunit of RNAPH and OmpR or some other transcription activators were described [31][32][33][34][35] . To elucidate the initiation steps for transcription activation by PmrA, the interactions between PmrA-DNA and the s 4 or C-terminal domain of the a-subunit of RNAPH remain to be characterized, which is in progress.…”