Trans-dominant mutations in the 3'-terminal region of the rpoB gene define highly conserved, essential residues in the beta subunit of RNA polymerase: the GEME motif.
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 alpha2betabeta'sigma. The beta subunit is conserved and has been implicated in all stages of transcription. The extreme C-terminus of the beta 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 C-terminus of the beta subunit of E. coli RNA polymerase. This involves random, PCR (Polymerase Chain Reaction)-mediated mutagenesis of the 3' region of rpoB encoding the C-terminal 116 amino acids of beta, followed by the isolation and characterization of trans-dominant-negative mutations.


RESULTS
Substitutions of conserved residues in this region were obtained that exhibited different degrees of growth inhibition in a host expressing the chromosomal-encoded wild-type form of the beta subunit. A number of different substitutions were isolated within the highly conserved sequence motif GEME (residues 1271-->1274 of the E. coli beta subunit). In addition, substitutions were obtained in the extreme C-terminal (surface-exposed) region of beta and at two residues previously proposed to be in the active site (H1237, K1242). The properties of the purified mutant holoenzymes, assessed by transcription assays in vitro, suggested a promoter blockading action.


CONCLUSIONS
We have identified an important, highly conserved motif in the beta subunit, GEME (residues 1271-->1274). The nature and effect of the amino acid substitutions at the Gly residue in GEME emphasize the importance of a small, uncharged residue at this position. The in vitro properties of the most extreme trans dominant-negative mutants altered in the GEME motif (and the mutant characteristics in vivo) were similar to those of certain previously identified active-site mutants, suggesting that the altered RNA polymerases were capable of promoter binding and RNA chain initiation but were deficient in the subsequent transcriptional stage.
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic