A 190 amino acid-long region centered around position 1050 of the 1407-amino acid-long  subunit of Escherichia coli RNA polymerase (RNAP) is absent from homologues in eukaryotes, archaea and many bacteria. In chloroplasts, the corresponding region can be more than 900 amino acids long. The role of this hypervariable region was studied by deletion mutagenesis of the cloned E. coli rpoC, encoding . Long deletions mimicking  from Gram-positive bacteria failed to assemble into RNAP. Mutants with short, 40 -60-amino acid-long deletions spanning  residues 941-1130 assembled into active RNAP in vitro. These mutant enzymes were defective in the transcript cleavage reaction and had dramatically reduced transcription elongation rates at subsaturating substrate concentrations due to prolonged pausing at sites of transcriptional arrest. Binding of a monoclonal antibody, Pyn1, to the hypervariable region inhibited transcription elongation and intrinsic transcript cleavage and, to a lesser degree, GreB-induced transcript cleavage, but did not interfere with GreB binding to RNAP. We propose that mutations in and antibody binding to the hypervariable, functionally dispensable region of  inhibit transcript cleavage and elongation by distorting the flanking conserved segment G in the active center.DNA-dependent RNA polymerases from eubacteria share a common subunit composition (1). The core RNAP 1 enzyme (subunit composition ␣ 2 Ј) is catalytically proficient but is unable to initiate transcription on promoters. Binding of a subunit converts the core enzyme into a holoenzyme, which can recognize a specific set of promoters (2). The  and Ј subunits together constitute more than 80% of the core RNAP mass and jointly form the catalytic center of the enzyme (3, 4). RNAPs from eukaryotes and archaea have subunits that are homologous to  and Ј of eubacterial enzymes (5-7). The evolutionary conservation within the /Ј lineages is limited to relatively short segments of primary sequence; each subunit has 8 -10 highly conserved segments. The amino-to carboxyl-terminal order of the conserved segments is invariant.The spacing between the conserved segments can vary even when subunits from closely related species are compared, due to an accumulation of insertions and deletions. There are several reasons why studies of such evolutionarily variable regions can shed light on RNAP structure and function. First, these regions may form docking sites for species-specific regulators of transcription (8, 9). Second, variable regions are likely to be surface-exposed and can therefore be used for affinity tagging of RNAP and transcription complexes (10). Third, variable regions often tolerate splits, allowing preparation of functional RNAP with relatively short  and/or Ј subunit fragments, dramatically facilitating mapping of protein-protein and protein-nucleic acid contacts during transcription (11,12).The focus of this report is an evolutionary hypervariable region in the C-terminal portion of Escherichia coli Ј (amino acids 1141-1131)...
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...
SummaryThe genes encoding the b-and b8-subunits of RNA polymerase (rpoB and rpoC respectively) are fused as one continuous open reading frame in Helicobacter pylori and in other members of this genus, but are separate in other bacterial taxonomic groups, including the closely related genus Campylobacter. To test whether this b±b8 tethering is essential, we used polymerase chain reaction-based cloning to separate the rpoB and rpoC moieties of the H. pylori rpoB ±rpoC fusion gene with a non-polar chloramphenicol resistance cassette containing a new translational start, and introduced this construct into H. pylori by electrotransformation. H. pylori containing these separated rpoB and rpoC genes in place of the native fusion gene produced non-tethered b and b8 RNAP subunits, grew well in culture and colonized and proliferated well in conventional C57BL/6 mice. Thus, the extraordinary b±b8 tethering is not essential for H. pylori viability and gastric colonization.
The genes coding for the β (rpoB) and β′ (rpoC) subunits of RNA polymerase are fused in the gastric pathogen Helicobacter pylori but separate in other taxonomic groups. To better understand how the unique fused structure evolved, we determined DNA sequences at and around therpoB-rpoC junction in 10 gastric and nongastric species ofHelicobacter and in members of the related generaWolinella, Arcobacter,Sulfurospirillum, and Campylobacter. We found the fusion to be specific to Helicobacter andWolinella genera; rpoB and rpoCoverlap in the other genera. The fusion may have arisen by a frameshift mutation at the site of rpoB and rpoC overlap. Loss of good Shine-Dalgarno sequences might then have fixed the fusion in the Helicobacteraceae, even if fusion itself did not confer a selective advantage.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.