Mechanism of initiation of transcription by Bacillus subtilis RNA polymerase at several promoters

Whipple, Frederick W.; Sonenshein, Abraham L.

doi:10.1016/0022-2836(92)90660-c

Cited by 76 publications

(69 citation statements)

References 54 publications

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“…The formation of open complexes at most E. coli promoters is essentially irreversible (25). In contrast, at the majority of promoters, B. subtilis enzyme forms unusually unstable open complexes that are in equilibrium with closed complexes and, in turn, with free RNAP (12,57,69). Consequently, B. subtilis core RNAP complexed with its major factor ( A ) utilizes E. coli promoters poorly (26,46), in spite of the essential identity of the Ϫ35 and Ϫ10 consensus promoter elements recognized by A and its E. coli homolog, 70 (23,24).…”

Section: Resultsmentioning

confidence: 99%

“…For several reasons, we think that these interactions could account for many differences between the B. subtilis and E. coli enzymes during transcription. First, altered contacts to the downstream DNA duplex could explain why, unlike E. coli RNAP, Bacillus enzyme is unable to protect the downstream DNA from the DNase I digestion (49,69) and forms unstable open complexes at many promoters (12,57,69). Substitutions that confer open complex instability in the E. coli enzyme (5, 59, 73) are located not only in the downstream clamp, which interacts with the duplex DNA directly (30), but also in the rifampin-binding pocket that interacts with the RNA-DNA hybrid (30).…”

Section: Discussionmentioning

confidence: 99%

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RNA Polymerases from Bacillus subtilis and Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

Artsimovitch¹,

Svetlov

Anthony

et al. 2000

J Bacteriol

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Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the ␤ and ␤ subunits and the downstream DNA duplex.Live bacteria are found in a variety of habitats, ranging from underwater volcano craters to arctic seas to the interior of plant and animal cells. The ability of bacterial species to thrive in an extraordinary wide range of conditions depends on diverse molecular mechanisms that adjust gene expression patterns according to nutrient flux and fluctuations of macroscopic parameters such as temperature, pressure, and pH.DNA-dependent RNA polymerase (RNAP) is the main target for the regulation of gene expression and is composed of several subunits in all cellular organisms. Four subunits of eubacterial RNAP (␣ 2 ␤␤Ј) comprise the core enzyme that is capable of basic polymerization activity in vitro, but requires the specificity factor () to initiate transcription from a promoter (44, 65). Orthologs of bacterial core subunits have been identified in Archaea and Eucarya (35), suggesting conservation of the basic architecture and function of RNAPs in all forms of life.A substantial amount of mechanistic and structural information regarding transcription in bacteria has been accumulated, but most of this information is derived from only a few model organisms. The disparity between the bacterial diversity and the scope of detailed transcriptional analysis is quite staggering. Out of the estimated 10 6 to 10 9 free-living and parasitic bacterial species (13), studies of only three RNAPs, from Escherichia coli, Bacillus subtilis, and Thermus aquaticus, together account for most of the information accumulated in the field to date. Moreover, none of these three data sets is complete. The high-resolution structural information is available only for the T. aquaticus RNAP, which has not been studied mechanistically. In contrast, the E. coli enzyme has served as the model for the majority of the biochemical and biophysical studies in transcription but has proven refractory to structural analysis except for the fragments of its smallest, ␣ and , subunits (1...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

RNA Polymerases from Bacillus subtilis and Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

Artsimovitch¹,

Svetlov

Anthony

et al. 2000

J Bacteriol

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“…Based on these findings, we propose that B. subtilis Gre factor plays a similar role during the initiation of RNA synthesis in many promoters or promoter proximal regions. Although the resolution of our ChAP-chip analysis did not permit discrimination of RNAP accumulation at promoters or promoter-proximal regions, we favor the possibility of accumulation at promoter-proximal pausing, since B. subtilis RNAP is known to form unstable open complexes and synthesize smaller amounts of abortive transcripts than E. coli RNAP (2,40). Recently, it was reported that the pausing of RNAP in E. coli is induced by direct and sequence specific interactions of RNAP with promoter-like sequences (6,29).…”

Section: Discussionmentioning

confidence: 78%

Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase in Bacillus subtilis Cells

et al. 2011

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Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that Escherichia coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis (chromatin affinity precipitation coupled with DNA microarray) was conducted to visualize the distribution of Bacillus subtilis GreA on the chromosome and to determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of the enzyme (12). In eukaryotic cells, the transcription factor, TFIIS, exerts similar activity (9), indicating that Gre function is evolutionarily conserved in multisubunit RNAPs (9). Gre factors consist of an N-terminal extended coiled-coil domain (NTD) and C-terminal globular domain (CTD) (19,32). Escherichia coli possesses two highly homologous Gre factors: GreA and GreB. A structural study on the RNAP-GreB complex further revealed that CTD binds to the rim of the secondary channel of RNAP through which substrate nucleoside triphosphates for RNA synthesis enter the catalytic site (18,25,38), while NTD extends into the secondary channel and the tip reaches the catalytic center (28). Two acidic residues, D41 and E44, located at the tip of NTD, are conserved in Gre factors, including those of Bacillus subtilis, and are proposed to assist RNAP function by coordinating the Mg 2ϩ ion and water molecule required for catalysis of RNA hydrolysis (20,28,31).During the elongation process of transcription, roadblocks generated by DNA-binding proteins or specific DNA sequences induce RNAP to slide backward along the template (backtrack), resulting in extrusion of the 3Ј terminus of nascent RNA through the RNAP secondary channel (9). Several biochemical and genetic studies have confirmed that the Gre factor facilitates endonucleolytic cleavage of extruded RNA to generate a new terminus that can be extended by RNAP, thus preventing transcription arrest during elongation and enhancing t...

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“…E. coli RNA polymerase holoenzyme and core enzyme forms were purchased from Epicenter. B. subtilis RNA polymerase holoenzyme was prepared by F. W. Whipple (29). B. subtilis RNA polymerase core enzyme was a gift from J. Helmann (Cornell University, Ithaca, NY).…”

Section: Methodsmentioning

confidence: 99%

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

Mani

Dupuy²

2001

Proc. Natl. Acad. Sci. U.S.A.

226

250

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Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The level of toxin production appears to be critical for determining the severity of the disease, but the mechanism by which toxin synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate synthesis of toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of toxin genes.

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Mechanism of initiation of transcription by Bacillus subtilis RNA polymerase at several promoters

Cited by 76 publications

References 54 publications

RNA Polymerases from Bacillus subtilis and Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

RNA Polymerases from Bacillus subtilis and Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase in Bacillus subtilis Cells

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

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