Roles of RNA Polymerase Downstream Mobile Elements in Transcription Initiati

描述（由申请人提供）：RNA聚合酶（RNAP），长期以来被认为是催化模板化RNA合成的复杂分子机器，最近被证明也可以作为分子“异构化”机器来制备启动子DNA和RNAP本身，以启动RNA合成。上游DNA在RNAP“背面”的弯曲和包裹使其能够与下游可移动元件（DME）相互作用；这种相互作用需要将其他二甲醚移出活性位点裂缝，并允许RNAP将下游双工DNA弯曲到裂缝中以有效打开。打开后，这些相同的二甲醚在步骤中组装成一个稳定的结构，似乎包围了下游的双相，形成了一系列在稳定性和寿命上有很大不同的开放复合物。这些开放复合物中的模板链似乎位于活性位点；裂口中非模板链下游部分的重新定位，可能是由图70的ssDNA模拟区1.1促成的，伴随着DME的这些构象变化。据报道，二甲醚的大构象变化也发生在起始的第一步。我们的长期目标是确定DME在启动子DNA和RNAP的这些大规模构象变化中的作用，这些构象变化是初始启动子识别（封闭）复合体转化（经典称为“异构化”）到具有起始能力的开放复合体，以及随后的转录起始和向延伸过渡所需要的。具体目标包括：确定下游移动元件（DME）在异构化的早期步骤中的功能，这些步骤包裹上游DNA，将下游双链DNA置于裂缝中，并通过表征DME缺失对这些步骤的动力学和关键中间封闭复合物I1结构的影响来打开它。2)确定二甲醚在初始不稳定开配合物（I2）向稳定RPo转化中的作用。3)确定二甲醚在起始催化步骤中的功能，并验证在LPR上检测到的三种开放配合物（I2、I3、RPo）在结构和功能上与rrnB P1、T7A1和LPR等三类开放启动物类似的假设。研究人员建议通过实验来确定WT和关键DME缺失的变体RNAP在异构化速率、开放配合物性质、起始速率以及DksA响应方面差异的结构和机制起源。使用的方法包括足迹法、荧光法（FRET、猝灭法）和瞬态（不稳定）闭合和开放配合物的交联研究，以表征它们以及与它们相关的决定速率的打开步骤。此外，不同的开放配合物及其假定的DME的不同组装状态将使用溶质探针，足迹和生产/流产引发分析来表征。

DESCRIPTION (provided by applicant): RNA polymerase (RNAP), long known as a sophisticated molecular machine in catalysis of templated RNA synthesis, has recently been shown to also function as a molecular "isomerization" machine to prepare both promoter DNA and RNAP itself for initiation of RNA synthesis. Bending and wrapping of the upstream DNA on the "back" side of RNAP positions it to interact with downstream mobile elements (DME); this interaction is required to move other DME out the active site cleft and allow RNAP to bend downstream duplex DNA into the cleft for efficient opening. After opening, these same DME assemble in steps into a stabilizing structure that appears to encircle the downstream duplex, creating a series of open complexes that differ greatly in stability and lifetime. The template strand in these open complexes appears to be positioned in the active site; repositioning of the downstream portion of the nontemplate strand in the cleft, possibly facilitated by the ssDNA mimic region 1.1 of ¿70, accompanies these conformational changes in the DME. Large conformational changes in DME are reported to occur in the first steps of initiation as well. Our long term goal is to determine the roles of the DME in these large-scale conformational changes in promoter DNA and RNAP that are needed for conversion (classically called "isomerization") of the initial promoter recognition (closed) complex to initiation- capable open complexes, and subsequently for transcription initiation and the transition to elongation. Specific aims include: ) Determine the functions of downstream mobile elements (DME) in the early steps of isomerization that wrap upstream DNA, place downstream duplex DNA in the cleft, and open it by characterizing effects of DME deletions on the kinetics of these steps and on the structure of the key intermediate closed complex I1. 2) Determine the functions of the DME in the conversion of the initial unstable open complex (I2) to the stable RPo. 3) Determine the functions of the DME in catalytic steps of initiation, and test the hypothesis that the three open complexes detected at LPR (I2, I3, RPo) are structural and functional analogs of the three classes of open promoter complexes exemplified by rrnB P1, T7A1, and LPR. Experiments are proposed to determine the structural and mechanistic origins of differences in isomerization rate, in properties of open complexes, in initiation rate, and in response to DksA between WT and variant RNAP with deletions in key DME. Methods to be used include footprinting, fluorescence (FRET, quenching assays) and crosslinking studies of transient (unstable) closed and open complexes to characterize them and the rate-determining opening step that relates them. In addition the different open complexes and their putative different states of assembly of the DME will be characterized using solute probes, footprinting, and productive/abortive initiation assays. PUBLIC HEALTH RELEVANCE: Research into the roles of downstream mobile elements in transcription initiation by E. coli RNA polymerase is needed to design novel classes of antibiotics that specifically inhibit bacterial transcription. Although the genomes of many disease-causing bacteria are now known, information regarding regulation of transcription in these organisms is completely lacking. Because the architecture and sequence of bacterial RNAP are highly conserved, this work provides a starting point for understanding the regulation of initiation of gene expression in other bacteria, with particular emphasis on virulent gene pathways.