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。     

Protein-induced DNA bending clarifies the architectural organization of the sigma54-dependent glnAp2 promoter

Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.     

Coupling σ Factor Conformation to RNA Polymerase Reorganisation for DNA Melting

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (σ⁵⁴-RNAP, Eσ⁵⁴) and a slowly hydrolysed ATP analogue (ATPγS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of σ⁵⁴-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Eσ⁵⁴ occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity σ⁵⁴ factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic β/β′ subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Eσ⁵⁴ promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the β′ jaw domain, thereby allowing acquisition of the open complex status.     

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.