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

• Published in: Genes to cells : devoted to molecular & cellular mechanisms Vol. 4; no. 3; pp. 145 - 159
• Main Authors: Cromie, Karen D., Ahmad, Khalid, Malik, Talat, Buyukuslu, Nihal, Glass, Robert E.
• Format: Journal Article
• Language: English
• Published: Oxford BSL Blackwell Science Ltd 01.03.1999
• Subjects: Amino Acid Sequence; Conserved Sequence; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; Genes, Bacterial; Molecular Sequence Data; Mutagenesis, Insertional; Phenotype; Plasmids; Polymerase Chain Reaction - methods; Structure-Activity Relationship; Transcription, Genetic
• ISSN: 1356-9597; 1365-2443
• DOI: 10.1046/j.1365-2443.1999.00248.x

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 α2ββ′σ. The β subunit is conserved and has been implicated in all stages of transcription. The extreme C‐terminus of the β 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 β 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 β, 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 β subunit. A number of different substitutions were isolated within the highly conserved sequence motif GEME (residues 1271→1274 of the E. coliβ subunit). In addition, substitutions were obtained in the extreme C‐terminal (surface‐exposed) region of β 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 β 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.