Different thermal energy requirement for open complex formation by<i>Escherichia coli</i>RNA polymerase at two related promoters

Grimes, Eileen A.; Busby, Stephen J. W.; Minchin, Stephen

doi:10.1093/nar/19.22.6113

Cited by 25 publications

(48 citation statements)

References 31 publications

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“…These observations support the idea that the TG motif sets a limit on the conformational fluctuation of the Ϫ10 region (34,40,41). This is consistent with analysis of the temperature dependence of promoter opening at galP1 and supports a mechanism of open complex formation whereby melting nucleates around Ϫ10 (42,43). Such a feature would be of particular importance at extended Ϫ10 promoters that lack an identifiable Ϫ35 hexamer.…”

Section: Discussionsupporting

confidence: 85%

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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Section: Discussionsupporting

confidence: 85%

Organization of Open Complexes at Escherichia coliPromoters

Bown

Owens

Meares

et al. 1999

Journal of Biological Chemistry

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“…The reason for this unusual behavior is not completely understood. For example, while it is clear that the extended Ϫ10 motif contributes to promoter opening at low temperature (9), it is not sufficient, since open promoter complexes on some extended Ϫ10 promoters are sensitive to low temperature (12,13).Previous work attempted to compare promoter complexes formed on the Ϫ10/Ϫ35 class and the extended Ϫ10 class promoters (9,12,14). However, due to technical constrains, relatively large fragments of promoter DNA were altered.…”

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confidence: 99%

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On the Role of the Escherichia coli RNA Polymerase σ70 Region 4.2 and α-Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter

Minakhin

Severinov

2003

Journal of Biological Chemistry

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Bacterial promoters of the extended ؊10 class contain a single consensus element, and the DNA sequence upstream of this element is not critical for promoter activity. Open promoter complexes can be formed on an extended ؊10 Escherichia coli galP1 promoter at temperatures as low as 6°C, when complexes on most promoters are closed. Here, we studied the contribution of upstream contacts to promoter complex formation using galP1 and its derivatives lacking the extended ؊10 motif and/or containing the ؊35 promoter consensus element. promoter sequences permit the derivation of consensus sequences for the Ϫ10 and Ϫ35 promoter elements and show that most promoters deviate from the consensus (3). Promoter elements of strong promoters tend to deviate from consensus less than promoter elements of weak promoters. Thus, assuming that promoter elements with consensus sequence are preferred binding sites for 70 regions 2.4 and 4.2, the strength of regions 2.4 and 4.2 interaction with their respective promoter elements determines the efficiency of promoter complex formation.For most promoters, RNAP 70 regions 2.4 and 4.2 interactions with their target promoter elements are sufficient for promoter complex formation. On some promoters, the presence of RNAP ␣-subunit C-terminal domains (␣CTDs) greatly increases transcription initiation beyond the basal level achieved through 70 -promoter element interactions (4). On these promoters, ␣CTDs make sequence-specific interactions with an A-rich promoter element (the "UP-element") located upstream of the Ϫ35 promoter element (reviewed by Gourse et al., Ref. 5). In the absence of a UP-element, ␣CTD non-specifically interacts with upstream DNA and the stimulatory effect of these interactions is less significant.There exists a minor class of promoters that lack recognizable Ϫ35 promoter elements and whose Ϫ10 elements are extended with an upstream dinucleotide TG. Genetic data show that specific interaction between an additional region of 70 , conserved region 2.5, and the TG motif is required for promoter complex formation on promoters of this class (6). Evidently, this additional contact is strong enough to make promoter complex formation on extended Ϫ10 promoters independent of 70 region 4.2 and Ϫ35 promoter element interaction (7).In order for template-directed RNA synthesis to occur, promoter DNA has to become locally melted (opened). In the catalytically competent open promoter complex, the melting extends from Ϫ12 to ϩ3 positions and thus includes the entire Ϫ10 promoter element. Promoter opening is temperature-dependent, and promoter complexes formed on Ϫ10/Ϫ35 type promoters "close" below 15°C (8). In contrast, promoter complexes on the extended Ϫ10 galP1 promoter remain open at temperatures as low as 5°C (9 -12). The reason for this unusual behavior is not completely understood. For example, while it is clear that the extended Ϫ10 motif contributes to promoter opening at low temperature (9), it is not sufficient, since open promoter complexes on some extended Ϫ10 promoters are sen...

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“…2 The open complex between σ 70 subunit and promoter elements, in which the promoter DNA is locally melted, is competent to initiate RNA synthesis according to the DNA sequence of template strand. 3,4 Promoter opening is temperature-dependent; the most −10/−35 promoter complexes display closed forms below 15 o C, 5 whereas promoter complexes on the extended −10 galP1 promoter remain open at 5 o C. 6,7 The reason for this unusual behavior of the −10 galP1 promoter is not completely understood.…”

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confidence: 99%