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Abstract
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 degrees 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. A panel of E. coli RNA polymerase holoenzymes containing two, one, or no alpha-subunit C-terminal domains (alpha CTD) and either wild-type sigma 70 subunit or sigma 70 lacking region 4.2 was assembled and tested for promoter complex formation. At 37 degrees C, alpha CTD and sigma 70 region 4.2 were individually dispensable for promoter complex formation on galP1 derivatives with extended -10 motif. However, no promoter complexes formed when both alpha CTD and sigma 70 region 4.2 were absent. Thus, in the context of an extended -10 promoter, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA can functionally substitute for each other. In contrast, at low temperature, alpha CTD and sigma 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for sigma 70 region 4.2 but not alpha CTD was required for open promoter complex formation on galP1 derivatives with extended -10 motif. We propose a model involving sigma 70 region 4.2 interaction with the beta flap domain that explains these observations.
Highlights
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)
At low temperature, ␣CTD and 70 region 4.2 interactions with upstream DNA were found to be functionally distinct, for 70 region 4.2 but not ␣CTD was required for open promoter complex formation on galP1 derivatives with extended ؊10 motif
Several conclusions about the galP1 promoter complex formation by E. coli RNAP 70 holoenzymes can be drawn from the experiments presented
Summary
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). 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). 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). Most Escherichia coli promoters are characterized by the presence of two 6-bp sequence elements centered ϳ10 and 35 nucleotides upstream of the transcription initiation point These promoters are referred to as Ϫ10/Ϫ35 class promoters. Interaction of the RNA polymerase (RNAP)1 70 subunit with Ϫ10 and Ϫ35 promoter elements is responsible for promoter recognition and transcription initiation. 70 conserved region 4.2 recognizes the Ϫ35 promoter element, while 70 conserved region 2.4 recognizes the Ϫ10 promoter element
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