A structure/function analysis of<i>Escherichia coli</i>RNA polymerase

Gross, Carol A.; Chan, Cathleen L.; Lonetto, Michael A.

doi:10.1098/rstb.1996.0045

Cited by 42 publications

(15 citation statements)

References 54 publications

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“…RpoS is the sigma factor most closely related to the vegetative RpoD ( 70 ) (28). The two sigmas recognize very similar promoter sequences, which is reflected in a high degree of homology in their 2.4 regions (Fig.…”

Section: Isolation Of Mutations In the Putative Turnover Element Inmentioning

confidence: 99%

Regulation of RpoS proteolysis in Escherichia coli : The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

Becker

Klauck

Hengge‐Aronis

1999

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

177

209

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The degradation of the RpoS ( S ) subunit of RNA polymerase in Escherichia coli is a prime example of regulated proteolysis in prokaryotes. RpoS turnover depends on ClpXP protease, the response regulator RssB, and a hitherto uncharacterized ''turnover element'' within RpoS itself. Here we localize the turnover element to a small element (around the crucial amino acid lysine-173) directly downstream of the promoter-recognizing region 2.4 in RpoS. Its sequence as well as its location identify the turnover element as a unique proteolysis-promoting motif. This element is shown to be a site of interaction with RssB. Thus, RssB is functionally unique among response regulators as a direct recognition factor in ClpXP-dependent RpoS proteolysis. Binding of RssB to RpoS is stimulated by phosphorylation of the RssB receiver domain, suggesting that environmental stress affects RpoS proteolysis by modulating RssB affinity for RpoS. Initial evidence indicates that lysine-173 in RpoS, besides being essential of RpoS proteolysis, may play a role in promoter recognition. Thus the same region in RpoS is crucial for proteolysis as well as for activity as a transcription factor. RpoS orS is a sigma subunit of RNA polymerase that is present at very low levels in exponentially growing Escherichia coli cells. In response to various stress conditions, RpoS is strongly up-regulated and activates 50-100 genes, which results in multiple stress resistance and other physiological and morphological alterations (for recent reviews, see refs. 1 and 2). The control of the cellular RpoS content occurs at the levels of rpoS transcription and translation as well as RpoS proteolysis. In exponentially growing cells, RpoS is a very unstable protein (with a half-life of approximately 2 min), but RpoS is stabilized in response to carbon starvation or shift to high osmolarity, high temperature, or low pH (3-7).Some trans-acting factors involved in the control of RpoS proteolysis have been described. The relevant protease is ClpXP (8), a complex ATP-dependent protease consisting of proteolytic (ClpP) and chaperone (ClpX) subunits that form a proteasome-like assembly (9, 10). In addition, a twocomponent-type response regulator, RssB (also termed SprE or MviA), is essential for RpoS degradation (3,11,12). The C-terminal output domain of RssB is unlike that of any other response regulator and also does not show similarity to other proteins of known function. So far, its molecular function has remained unknown. RpoS degradation in vivo is positively modulated by acetyl phosphate, which readily phosphorylates the D58 residue in the RssB receiver domain in vitro (13).In addition to these trans-acting factors, a ''turnover element'' within RpoS is required for its proteolysis. The turnover element confers instability upon other proteins, e.g., RpoS-␤-galactosidase hybrid proteins (5,8). Thus, it may be functionally comparable to proteolysis-promoting elements in various eukaryotic proteins, such as the ''destruction box '' or D-box (14). The exact loca...

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Section: Isolation Of Mutations In the Putative Turnover Element Inmentioning

confidence: 99%

Regulation of RpoS proteolysis in Escherichia coli : The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

Becker

Klauck

Hengge‐Aronis

1999

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

177

209

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“…Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of 70 contact the Ϫ10 and Ϫ35 hexamers, respectively (1,2), whereas the C-terminal domain of the ␣ subunit (␣CTD) contacts the UP element (4). Recent work from this laboratory has indicated that a newly defined region of 70 , region 2.5, is responsible for making sequence-specific contacts with the extended Ϫ10 motif (12).…”

mentioning

confidence: 99%

“…E. coli uses a single core RNA polymerase for transcription elongation with subunit composition ␣ 2 ␤␤Ј. Promoter specificity is principally afforded by a separate subunit, , which associates with the core enzyme to give holoenzyme (RNAP) 1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The 70 subunit, encoded by rpoD, is one of several subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth.…”

mentioning

confidence: 99%

“…RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of 70 to make stable contacts with the promoter DNA (1,2). E. coli promoters contain two very conserved motifs, the Ϫ10 and Ϫ35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended Ϫ10 motif (5).…”

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confidence: 99%

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Organization of Open Complexes at Escherichia coliPromoters

Bown

Owens

Meares

et al. 1999

Journal of Biological Chemistry

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A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of 70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered 70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA⅐Fe (FeBABE). RNA polymerase core reconstituted with tagged derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the ؊10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of with promoter DNA is discussed.Promoter recognition requires sequence-specific contacts by the transcriptional apparatus. At most promoters these contacts are made upstream from the transcription start point. Once the transcriptional apparatus has bound the promoter to form a closed complex, an isomerization event occurs to generate the open complex, forming the single-stranded template required for transcription. The bacterium Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription elongation with subunit composition ␣ 2 ␤␤Ј. Promoter specificity is principally afforded by a separate subunit, , which associates with the core enzyme to give holoenzyme (RNAP) 1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The 70 subunit, encoded by rpoD, is one of several subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth. RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of 70 to make stable contacts with the promoter DNA (1, 2). E. coli promoters contain two very conserved motifs, the Ϫ10 and Ϫ35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended Ϫ10 motif (5). The extended Ϫ10 motif (5Ј-TGXTATAAT-3Ј) can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within the Ϫ35 region (6, 7). Therefore this TGX motif is able to compensate for a poor Ϫ35 hexamer. The TG motif has been shown to be important for promoter activity in several other bacterial species (8 -11). Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of 70 contact the Ϫ10 and Ϫ35 hexamers, respectively (1, 2), whereas the C-terminal domain of the ␣ subunit (␣CTD) contacts the UP element (4). Recen...

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“…The latter, being heterotetramers of the composition (␣) 2 ␤␤Ј, resemble eucaryal RNA polymerases in their overall appearance (3)(4)(5)(6) and in the structural core composition, with the two largest procaryal subunits, ␤ and ␤Ј, having fairly conserved eucaryal orthologs, represented by yeast Rpb1 and Rpb2 (reviewed in Ref. 7). Less obvious homology was noted between eubacterial ␣ subunit and yeast Rpb3 (8), consistent with the reported functional equivalence of ␣ 2 and (Rpb3) 2 dimers in assembly of their respective enzymes (9).…”

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confidence: 99%

Rpb3, Stoichiometry and Sequence Determinants of the Assembly into Yeast RNA Polymerase II in Vivo

Svetlov

Nolan

Burgess

1998

Journal of Biological Chemistry

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Stoichiometry of the third largest subunit (Rpb3) of the yeast RNA polymerase II is a subject of continuing controversy. In this work we utilized immunoaffinity and nickel-chelate chromatographic techniques to isolate the RNA polymerase II species assembled in vivo in the presence of the His 6 -tagged and untagged Rpb3. The distribution pattern of tagged and untagged subunits among the RNA polymerase II molecules is consistent with a stoichiometry of 1 Rpb3 polypeptide per molecule of RNA polymerase. Deletion of either ␣-homology region (amino acids 29 -55 or 226 -267) from the Rpb3 sequence abolished its ability to assemble into RNA polymerase II in vivo.Yeast Saccharomyces cerevisiae RNA polymerase II is a multisubunit enzyme comprised of 12 core polypeptides (1). With minor variations its subunit composition is characteristic of all eucaryal nuclear RNA polymerases and their archaeal counterparts (1, 2). Despite the trivial quantitative differences, a profound similarity can also be found between these eucaryal/ archaeal multisubunit enzymes and eubacterial RNA polymerases. The latter, being heterotetramers of the composition (␣) 2 ␤␤Ј, resemble eucaryal RNA polymerases in their overall appearance (3-6) and in the structural core composition, with the two largest procaryal subunits, ␤ and ␤Ј, having fairly conserved eucaryal orthologs, represented by yeast Rpb1 and Rpb2 (reviewed in Ref. 7). Less obvious homology was noted between eubacterial ␣ subunit and yeast Rpb3 (8), consistent with the reported functional equivalence of ␣ 2 and (Rpb3) 2 dimers in assembly of their respective enzymes (9).Young and co-authors also concluded, based on the [S 35 ]Met labeling of the RNA polymerase II subunits, that two copies of the subunits Rpb3, Rpb5, and Rpb9 were present in each RNA polymerase II molecule, whereas the rest were represented by a single polypeptide each or else recovered in submolar amounts (10). These inferred orthological relations between ␣ 2 and (Rpb3) 2 dimers were later questioned by the reports from several laboratories, that failed to detect homodimerization potential in yeast Rpb3 and its higher eucaryal homologs (11-13). Instead, in an array of in vitro assays, Rpb3 was shown to associate with another apparent ␣-subunit homolog, Rpb11, with a stoichiometry of 1:1 (11, 13). Human homologs of Rpb3 and 11 were also shown to associate in a complex of unknown stoichiometry in the yeast two-hybrid system (12). It was consequently suggested that the Rpb3-Rpb11 heterodimer serves as eucaryal analog of the ␣ 2 -homodimer (11); consistent with this hypothesis is the recovery of a Rpb2-Rpb3-Rpb11 core subassembly from a partially denatured Schizosaccharomyces pombe RNA polymerase II (14).In this work we utilized an independent method to ascertain the stoichiometry of the yeast Rpb3, based on simultaneous expression of the wild-type (genomic) RPB3 gene and its plasmid-borne His 6 -tagged version followed by affinity purification of the in vivo assembled RNA polymerase II species. EXPERIMENTAL PROCEDURESP...

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A structure/function analysis ofEscherichia coliRNA polymerase

Cited by 42 publications

References 54 publications

Regulation of RpoS proteolysis in Escherichia coli : The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

Regulation of RpoS proteolysis in Escherichia coli : The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

Organization of Open Complexes at Escherichia coliPromoters

Rpb3, Stoichiometry and Sequence Determinants of the Assembly into Yeast RNA Polymerase II in Vivo

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