Roles of RNA Polymerase Downstream Mobile Elements in Transcription Initiati

RNA 聚合酶下游移动元件在转录起始中的作用

• 批准号: 8539635
• 负责人: M. THOMAS RECORD
• 金额: $ 27.15万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-03 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539635
• 关键词: Active Sites; Antibiotics; Architecture; Back; Bacteria; Bacterial RNA; Beryllium; Biological Assay; Catalysis; Cleaved cell; Communication; Complex; Coupled; DNA; DNA-Directed RNA Polymerase; Disease; Elements; Escherichia coli; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Indium; Jaw; Kinetics; Methods; Molecular; Molecular Machines; Organism; Pathway interactions; Positioning Attribute; Process; Property; RNA chemical synthesis; Regulation; Reporting; Research; Role; Series; Side; Structure; Testing; Transcription Initiation; Transcriptional Regulation; Variant; Virulent; Work; analog; crosslink; design; novel; promoter; research study; response; solute

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.

描述（由适用提供）：RNA聚合酶（RNAP），较长地称为模板RNA合成催化中的复杂分子机，最近已被证明还可以作为分子“异构化”机器，以准备启动子DNA和RNAP本身以启动RNA合成。 RNA“背面”侧上游DNA的弯曲和包裹定位与下游移动元件相互作用（DME）；需要这种相互作用才能将其他DME移出活跃的位点裂缝，并使RNAP将下游双链DNA弯曲到裂缝中以有效开放。打开后，这些相同的DME在台阶中组装成一个稳定结构，该结构似乎环绕着下游双链体，从而产生了一系列的开放式复合物，在稳定性和寿命方面有所不同。这些开放式复合物中的模板链似乎位于活动位置。重新定位裂缝中非板岩链的下游部分，这可能是由ssDNA模拟区1.1的70的模拟区域制备的，涉及DME中的这些会议变化。据报道，DME的大规模会议变化也发生在开始的第一步。我们的长期目标是确定DME在这些启动子DNA和RNAP中这些大规模会议变化中的作用，这些变化是对初始启动子识别（封闭）识别（经典称为“异构化”）所需的，这些启动子识别（封闭）综合体为有能力的开放配合物，以及随后用于转录启动以及向延长的过渡。具体目的包括：）在异构化的早期步骤中确定下游移动元件（DME）的功能，该步骤包裹上游DNA，将下游双链DNA放在裂缝中，并通过表征DME缺失对这些步骤的动力学以及关键中间封闭式复合物I1的结构的效果来打开它。 2）确定DME在初始不稳定开放式复合物（I2）转换为稳定RPO中的功能。 3）确定DME在开始的催化步骤中的功能，并检验以下假设：LPR（I2，I3，RPO）检测到的三个开放式复合物是由RRNB P1，T7A1和LPR等三类开放启动子复合物的结构和功能类似物。提出了实验来确定异构化速率，开放式复合物，起始速率的差异的结构和机械起源，以及wt和变体RNAP之间的DKSA的响应，并在键DME中删除。要使用的方法包括足迹，荧光（FRET，淬火测定法）以及对瞬态（不稳定）闭合和开放式复合物的交联研究，以表征它们以及与它们相关的速率确定的开放步骤。此外，使用实心问题，足迹和生产性/流产的启动测定法，将对DME的不同开放式复合物及其假定的不同组装状态进行表征。