Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif

Abstract

Background: The multimeric DNA-dependent RNA polymerases are widespread throughout nature. The RNA polymerase of Escherichia coli, which is the most well characterized, consists of a holoenzyme with subunit stoichiometry of ␣ 2 ␤␤ 0 . The ␤ subunit is conserved and has been implicated in all stages of transcription. The extreme C-terminus of the ␤ subunit, which includes two well-conserved sequence segments, contributes to the active centre and has been proposed to act in transcriptional termination. We describe a genetic system for further characterizing the role of the extreme Cterminus of the ␤ subunit of E. coli RNA polymerase. This involves random, PCR (Polymerase Chain Reaction)-mediated mutagenesis of the 3 0 region of rpoB encoding the C-terminal 116 amino acids of ␤, followed by the isolation and characterization of trans-dominant-negative mutations.

Intragenic suppression of trans‐dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C‐terminus

“…Previous studies have defined the optimal conditions for the random mutagenesis of the 3 0 -116 codons, generating predominantly single base changes in the distal region of the ␤ gene (Cromie et al 1999). In brief, this entails the amplification by Taq DNA polymerase in the presence of 0.005 mM MnCl 2 .…”

“…The missense mutants are listed N→C terminal, and details of the initial, primary mutants are included (Cromie et al 1999). In column one, conserved residues are underlined, and 'ter' indicates a translational termination codon that results in a glutamine substitution owing to the presence of the supE suppressor in the host strain DH5␣.…”

“…4), ߛ/(ߛ) indicates strong/weak lac transcript production, respectively. Promoter blockading refers to the ability of purified mutant RNA polymerase, when preincubated under open promoter conditions, to inhibit the function of wild-type enzyme in a run-off assay (Cromie et al 1999). Determination of subunit assembly is described in the text; smallest polymerase component, and identified the (RNA polymerase-specific) polypeptides present with anti-holoenzyme antibodies.…”

“…We have previously described a genetic system involving random, PCR-mediated mutagenesis of the region of rpoB encoding the C-terminal 116 amino acids of the ␤ subunit of E. coli RNA polymerase, and the characterization of dominant-negative mutations (Cromie et al 1999). The rationale for this procedure was that we wished to isolate assembly-proficient mutants blocked in the transcriptional process as a means of identifying functionally important sites.…”

“…2). The substitutions are grouped according to their in vivo and in vitro action (class I mutants exhibiting a significantly greater effect than class II mutants) and further subdivided into those exhibiting promoter blockading in vitro (Cromie et al 1999). Conserved residues are emboldened (see also Fig.…”