Single Molecule Studies of Prokaryotic and Eukaryotic Transcription Machineries

原核和真核转录机制的单分子研究

基本信息

批准号：
8150994
负责人：
SHIMON WEISS
金额：
$ 36.5万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8150994
关键词：
Address Alkylation Anti-Bacterial Agents Bacterial RNA Biochemical Biological Assay Biological Models Complex Coupled Cysteine DNA DNA-Directed RNA Polymerase Diffuse Disease Drug Design Escherichia coli Fluorescence Fluorescence Microscopy Foundations Funding Gene Expression General Transcription Factors Genetic Transcription Health In Vitro Kinetics Knowledge Label Lasers Maps Mediating Messenger RNA Methods Microscopic Modeling Molecular Molecular Analysis Molecular Conformation Monitor Mutagenesis Nuclear Magnetic Resonance Pathway interactions Pharmaceutical Preparations Polymerase Preclinical Drug Evaluation Process Proteins Regulation Role Scanning Screening procedure Site Spectrum Analysis Staging Structure Therapeutic Agents Time Transcription Elongation Transcription Initiation Transcription Process Transcription-Coupled Repair Transcriptional Regulation Work X-Ray Crystallography base chemical cleavage conformational conversion design human disease insight novel prevent promoter protein protein interaction single molecule single-molecule FRET tool transcription factor transcription termination

项目摘要

DESCRIPTION (provided by applicant): This proposal seeks to unravel unresolved questions in the mechanisms and regulation of prokaryotic transcription initiation, elongation and termination, as well as in early stages of eukaryotic transcription initiation. Single molecule fluorescence spectroscopic and microscopic methods will be used to detail the sequence, magnitude, kinetics and order of conformational transitions and intermediates along the basal transcription cycle pathway. Transient protein-DNA and protein-protein interactions (and their induced conformations) of the prokaryotic transcription machinery's subunits and of some of the eukaryotic transcription machinery's subunits will be investigated. The mechanisms of prokaryotic and eukaryotic transcription regulation will also be explored using several transcription factors as model systems. Several single molecule assay formats, implemented on freely diffusing transcription complexes and on immobilized complexes will be utilized to address mechanistic and kinetic questions respectively. The proposed studies are expected to elucidate the role of conformational transitions and biomolecular interactions in transcription and its regulation, and the tools developed for these studies could be generalized for the study of regulatory circuits and other biomolecular machineries. The proposed studies of prokaryotic transcription will provide a basis for understanding antibacterial drugs (through inhibition of conformations) and therefore will provide relevant insight and assays for drugs design and screening. The proposed studies of eukaryotic transcription will lay a foundation for understanding mechanisms of transcription related human diseases and will aid in designing and screening therapeutics agents for these diseases. PUBLIC HEALTH RELEVANCE: The proposed work will unravel the detailed mechanism of the first and most important step in gene expression, i.e. transcription and transcription regulation. It will pave the way for detailed molecular and structural understanding of the action of antibacterial agents. It will also provide answers for several long standing and unresolved questions regarding the early stages of eukaryotic transcription initiation and regulation. The latter will provide detailed molecular understanding of the causes for transcription related diseases and will provide tools for drug design and screening that will prevent, manage and cure these diseases.

描述（由申请人提供）：该提案旨在阐明原核转录起始、延伸和终止的机制和调节以及真核转录起始的早期阶段中尚未解决的问题。单分子荧光光谱和显微方法将用于详细描述基础转录循环途径中构象转变和中间体的序列、幅度、动力学和顺序。将研究原核转录机器亚基和一些真核转录机器亚基的瞬时蛋白质-DNA 和蛋白质-蛋白质相互作用（及其诱导构象）。还将使用几种转录因子作为模型系统来探索原核和真核转录调控机制。在自由扩散的转录复合物和固定复合物上实施的几种单分子测定形式将分别用于解决机械和动力学问题。拟议的研究有望阐明构象转变和生物分子相互作用在转录及其调节中的作用，并且为这些研究开发的工具可以推广到调节回路和其他生物分子机制的研究。拟议的原核转录研究将为理解抗菌药物（通过构象抑制）提供基础，因此将为药物设计和筛选提供相关的见解和分析。拟议的真核转录研究将为理解转录相关人类疾病的机制奠定基础，并将有助于设计和筛选这些疾病的治疗剂。公共卫生相关性：拟议的工作将揭示基因表达的第一步也是最重要的一步的详细机制，即转录和转录调控。它将为详细了解抗菌剂作用的分子和结构铺平道路。它还将为有关真核转录起始和调控早期阶段的几个长期存在且未解决的问题提供答案。后者将提供对转录相关疾病原因的详细分子理解，并将提供用于预防、管理和治愈这些疾病的药物设计和筛选工具。