Structural basis of DNA recognition by the alternative sigma-factor, sigma54

The sigma subunit of bacterial RNA polymerase (RNAP) regulates gene expression by directing RNAP to specific promoters. Unlike sigma(70)-type proteins, the alternative sigma factor, sigma(54), requires interaction with an ATPase to open DNA. We present the solution structure of the C-terminal domain of sigma(54) bound to the -24 promoter element, in which the conserved RpoN box motif inserts into the major groove of the DNA. This structure elucidates the basis for sequence specific recognition of the -24 element, orients sigma(54) on the promoter, and suggests how the C-terminal domain of sigma(54) interacts with RNAP.     

Functional interaction between RNA polymerase alpha subunit C-terminal domain and sigma70 in UP-element- and activator-dependent transcription

We show that the Escherichia coli RNA polymerase (RNAP) alpha subunit C-terminal domain (alphaCTD) functionally interacts with sigma(70) at a subset of UP-element- and activator-dependent promoters, we define the determinants of alphaCTD and sigma(70) required for the interaction, and we present a structural model for the interaction. The alphaCTD-sigma(70) interaction spans the upstream promoter and core promoter, thereby linking recognition of UP-elements and activators in the upstream promoter with recognition of the -35 element in the core promoter. We propose that the alphaCTD-sigma(70) interaction permits UP-elements and activators not only to "recruit" RNAP through direct interaction with alphaCTD, but also to "remodel" RNAP-core-promoter interaction through indirect, alphaCTD-bridged interactions with sigma(70).     

Sigma54依赖性转录起始

作为细菌RNA聚合酶(RNAP)的组成型辅助因子,sigma70和sigma54分别参与了原核细胞不同类型基因的转录起始调控。sigma70负责管家基因的自发转录起始;而sigma54负责应激信号相关的基因转录起始。sigma54与RNAP形成复合物后,会通过空间阻滞的方式阻碍DNA进入RNAP中,抑制基因转录起始。当细胞环境变化后,特定应激信号会通过细菌增强子结合蛋白(bEBP)诱发sigma54的构象发生变化,解除sigma54对RNAP的抑制,启动sigma54依赖的基因转录。最近的结构生物学研究揭示了sigma54依赖性转录起始的若干复合物结构,包括全酶、封闭式复合物、2个中间状态复合物及开放式复合物。通过分析这些转录起始复合物的结构,本文阐述了转录起始过程中复合物的结构变化。描述并分析了sigma54和bEBP在转录起始过程中所发挥的功能。本文有助于了解转录起始分子水平的变化,为深入理解sigma54和bEBP促进转录起始的分子机制提供了参考。     

Inhibitory effect of phosphate on in vitro development of 2-cell rat embryos is overcome by a factor(s) in oviductal extracts

The promoter recognition site on the sigma70 initiation factor is shielded from interaction with DNA unless sigma70 is bound to the core component of RNA polymerase (RNAP). It is shown that interaction of sigma70 with the isolated beta' subunit of Escherichia coli RNAP is sufficient to induce unshielding of the DNA binding site. Using UV-induced DNA-protein cross-linking we demonstrate that free beta' stimulates specific cross-links between region 2 of the sigma70 polypeptide and a fragment of the non-template promoter strand containing the TATAAT sequence. Thus the sigmabeta' subassembly of RNAP can assume a functionally competent conformation independently of the bulk of the RNAP core.     

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.     

On the role of the Escherichia coli RNA polymerase sigma 70 region 4.2 and alpha-subunit C-terminal domains in promoter complex formation on the extended -10 galP1 promoter

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.