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，最近已经显示出也起分子“异构化”机器的作用，以制备启动子DNA和RNAP本身用于起始RNA合成。上游DNA在RNAP“背”侧的弯曲和包裹使其与下游移动的元件（DME）相互作用;需要这种相互作用将其他DME移出活性位点裂缝并允许RNAP将下游双链体DNA弯曲到裂缝中以有效打开。打开后，这些相同的DME逐步组装成一个稳定的结构，似乎包围下游的双链体，创造了一系列开放的复合物，在稳定性和寿命上有很大的不同。这些开放复合物中的模板链似乎位于活性位点;裂缝中非模板链下游部分的重新定位，可能由ssDNA模拟区域1.1的70促进，伴随着DME中的这些构象变化。据报道，DME中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中的这些大规模构象变化中的作用，所述大规模构象变化是初始启动子识别（封闭）复合物转化（经典地称为“异构化”）为能够起始的开放复合物以及随后转录起始和向延伸的过渡所需的。具体目标包括：通过表征DME缺失对这些步骤的动力学和对关键中间体封闭复合物I1的结构的影响，确定下游移动的元件（DME）在异构化的早期步骤中的功能，所述下游DME元件包裹上游DNA，将下游双链体DNA置于裂缝中，并将其打开。2)确定DME在初始不稳定开放复合物（I2）转化为稳定RPo中的作用。3)确定DME在引发的催化步骤中的功能，并检验在LPR处检测到的三种开放复合物（I2、I3、RPo）是以rrnB P1、T7 A1和LPR为例的三类开放启动子复合物的结构和功能类似物的假设。实验提出，以确定异构化率的差异，在开放的复合物的性质，在起始速率，并在响应WT和变异RNAP与关键DME中的缺失之间的DksA的结构和机制的起源。要使用的方法包括足迹法，荧光（FRET，淬灭试验）和交联研究的瞬态（不稳定）封闭和开放的复合物，以表征他们和速率决定开放的步骤，涉及他们。此外，不同的开放式复合物和它们的DME的推定的不同组装状态将使用溶质探针、足迹法和生产性/流产起始测定来表征。

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.