Roles of RNA Polymerase Downstream Mobile Elements in Transcription Initiati

描述(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。RNAP“背面”的上游DNA的弯曲和包裹使其能够与下游移动元件(DME)相互作用；这种相互作用是将其他DME移出活性部位裂隙并允许RNAP将下游双链DNA弯曲到裂隙中以实现有效打开所必需的。打开后，这些相同的DME逐步组装成稳定结构，似乎包围了下游的双链，产生了一系列在稳定性和寿命上大不相同的开放复合体。这些开放的复合体中的模板链似乎位于活性部位；非模板链的下游部分在裂隙中的重新定位，可能是由？70的ssDNA模拟区1.1促进的，伴随着DME中的这些构象变化。据报道，DME的大构象变化也发生在启动的第一步。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，这些变化是将最初的启动子识别(闭合)复合体转化(经典地称为“异构化”)为能够启动的开放复合体，以及随后的转录起始和向延伸的转变所必需的。具体目的包括：)确定下游移动元件(DME)在包裹上游DNA的异构化早期步骤中的功能，将下游双链DNA放置在裂隙中，并通过表征DME缺失对这些步骤的动力学和关键中间闭合复合体I1的结构的影响来打开裂隙。2)确定DME在初始不稳定的开放络合物(I2)转化为稳定的RPO过程中的作用。3)确定DME在催化引发步骤中的功能，并检验在LPR处检测到的三个开放复合体(I2、I3、RPO)是以rrnB P1、T7A1和LPR为代表的三类开放启动子复合体的结构和功能类似物的假设。实验被用来确定WT和带有关键DME缺失的RNAP变体在异构化速率、开放络合物的性质、引发速率以及对Dks A的响应方面存在差异的结构和机理根源。可使用的方法包括足迹、荧光(FRET、猝灭分析)和瞬时(不稳定)封闭和开放络合物的交联性研究，以表征它们以及与它们相关的决定速率的开放步骤。此外，不同的开放络合物及其可能的不同组装状态将使用溶质探针、足迹和生产/失败启动分析来表征。 与公共卫生相关：需要研究下游移动元件在大肠杆菌RNA聚合酶启动转录中的作用，以设计专门抑制细菌转录的新型抗生素。虽然许多致病细菌的基因组已为人所知，但关于这些生物体转录调控的信息完全缺乏。由于细菌RNAP的结构和序列高度保守，这项工作为理解其他细菌基因表达启动的调控提供了一个起点，特别是毒力基因途径。

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.