Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain

The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme. In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha. For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210. The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits. New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding. Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.     

Analysis of the beta' subunit of DNA-dependent RNA polymerase does not support the hypothesis inferred from 16S rRNA analysis that Oenococcus oeni (formerly Leuconostoc oenos) is a tachytelic (fast-evolving) bacterium

rRNA sequencing has shown that leuconostocs comprise three distinct phylogenetic lineages which have been designated separate genera (viz., the genera Leuconostoc sensu stricto, Oenococcus, and Weissella). In addition, the 16S rRNA line formed by Oenococcus oeni (formerly Leuconostoc oenos) is exceptionally long; this fact, together with variations in the compositions of conserved positions in the 16S rRNA, has led to the hypothesis (D. Yang and C. R. Woese, Syst. Appl. Microbiol. 12:145-149, 1989) that this organism is a fast-evolving bacterium. Previous evidence that the leuconostocs should be divided into three genera and that O. oeni is an example of tachytelic evolution has come solely from rRNA analyses. In this study we seqenced the rpoC gene encoding the beta' subunit of DNA-dependent RNA polymerase of leuconostocs and performed a comparative phylogenetic analysis. The subdivision of the leuconostocs into three distinct lineages was confirmed by the rpoC gene data, but no evidence that indicated that O. oeni is evolving at an extraordinary rate was found. If O. oeni is truly tachytelic, then fast-evolving phenomena would be expected to occur throughout the whole genome, including this independent molecular chronometer.     

Vasodilatory action of the calcium antagonist amlodipine on large and resistance pulmonary arteries from normoxic and chronically hypoxic rats

New findings are presented for the approximately 50 residue KH motif, a domain recently discovered in RNA-binding proteins. The conserved sequence is approximately 10 residues larger than previously reported. Profile searches have revealed new members of this family, including two, E. coli NusA and human GAP-associated p62 phosphoprotein, for which RNA-binding data exists. A nusA homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator. All KH-containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain.     

The largest subunits of RNA polymerase from gastric helicobacters are tethered

The rpoB and rpoC genes of eubacteria and archaea, coding respectively for the beta- and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed homologs of two genes in one continuous open reading frame that potentially could encode one 2890-amino acid-long beta-beta' fusion protein. Here, we show that this open reading frame does in fact encode a fused beta-beta' polypeptide. In addition, we establish by DNA sequencing that rpoB and rpoC are also fused in each of four other unrelated strains of H. pylori, as well as in Helicobacter felis, another member of the same genus. In contrast, the rpoB and rpoC genes are separate in two members of the related genus Campylobacter (Campylobacter jejuni and Campylobacter fetus) and encode separate RNA polymerase subunits. The Campylobacter genes are also unusual in overlapping one another rather than being separated by a spacer as in other Gram-negative bacteria. We propose that the unique organization of rpoB and rpoC in H. pylori may contribute to its ability to colonize the human gastric mucosa.