The Largest Subunits of RNA Polymerase from Gastric Helicobacters Are Tethered

Abstract

The rpoB and rpoC genes of eubacteria and archaea, coding respectively for the ␤-and ␤-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 ␤-␤ fusion protein.Here, we show that this open reading frame does in fact encode a fused ␤-␤ 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 Gramnegative 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. DNA-dependent RNA polymerase (RNAP)1 is the central enzyme of gene expression and a major target for regulation. RNAPs are large, multisubunit protein complexes. The best studied RNAP, from Escherichia coli (Ͼ400 kDa), contains four core polypeptides: ␤Ј (155 kDa), ␤ (150 kDa), a dimer of ␣ (37 kDa), and one of several possible (specificity) subunits. RNAPs from other bacteria have similar subunit composition and exhibit striking and co-linear sequence similarities with the E. coli enzyme (1). The two largest RNAP core subunits comprise 60% of the RNAP mass and appear to be responsible for most of the functions of the enzyme.The synthesis of RNAP subunits is coordinately regulated (2), but the exact mechanisms at play are unknown. In most eubacteria and archaea, genes encoding the ␤-and ␤Ј-like subunits are organized in an operon with the gene for the ␤-like subunit (rpoB) always preceding that for the ␤Ј-like subunit (rpoC) (3, 4). The two genes are separated by a short, untranslated linker (3, 5, 6), 2 whereas in archaea they overlap by several codons (4).The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed one continuous open reading frame containing the homologs of rpoB and rpoC, potentially encoding one fused 2890-amino acid-long ␤-␤Ј polypeptide (8). Our previous analysis using E. coli RNAP showed that such a ␤-␤Ј fusion is compatible with RNAP function: (i) the product of artificially fused rpoB and rpoC genes of E. coli could assemble into a functional RNAP in vivo and in vitro and (ii) an E. coli strain containing the fused rpoBC gene as its only source for RNAP was viable and contained RNAP of the expected (␤-␤Ј)␣ 2 subunit composition (9). This tethering of E. coli ␤ and ␤Ј increased the efficiency of RNAP assembly in vitro and suppressed an rpoC ts assembly mutation in vivo. 3 It thus seemed that natural tetherin...

N4 RNA Polymerase II, a Heterodimeric RNA Polymerase with Homology to the Single-Subunit Family of RNA Polymerases

“…Active E. coli RNAP has been assembled from ␤-subunit fragments (57). In Helicobacter pylori and Wolinella succinogenes, the ␤ and ␤Ј subunits of RNAP are naturally fused (68,69). Separation of the ␤ and ␤Ј subunits in H. pylori yields viable bacteria that are able to colonize mice (49).…”

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

“…The two archaeal ␤ homologues of the Euryarchaeota group are expressed as two 'half ' segments from separate cistrons, the B 0 protein carrying the three clusters (RPB1_HALHA and RPB1_METTH). Note that the homologue from Helicobacter pylori appears to be part of a ␤-␤ 0 fusion protein (Zakharova et al 1998; as initially reported for the putative hybrid ␤-␤ 0 polypeptide of the Kluyveromyces lactis killer plasmid, where the sequence is GQME rather than GEME; Wilson & Meacock 1988). trans-dominant mutants (G1271V and M1273V) on minimal rather than nutrient plates, where generation time is increased. Interestingly, all eight mutants obtained in this manner markedly alter protein structure (Table 1; see also Table 2): Three generate amber stop codons terminating translation close to the site of the original amino acid substitution (no such mutants were obtained on rich medium) while the other five mutants carry frameshifts.…”

Alanine-Threonine Polymorphism of Helicobacter pylori RpoB Is Correlated with Differential Induction of Interleukin-8 in MKN45 Cells

“…DNA-dependent RNA polymerase is a principal enzyme in the transcriptional process and of many regulatory pathways that control gene expression in living organisms. It is evolutionarily conserved in sequence, structure, and function from bacteria to humans (19,24,34). A high level of genetic diversity among H. pylori strains has also been observed by 16S ribosomal DNA sequence analysis (27).…”

“…rpoB encodes the ␤-subunit of RNA polymerase and is a highly conserved housekeeping gene. Comparisons of rpoB sequences have previously been used for phylogenetic analysis and for the differential identification of bacteria (14,15,19,34).…”