Single Molecule Studies of Prokaryotic and Eukaryotic Transcription Machineries

描述(申请人提供)：本提案旨在解开原核生物转录起始、延长和终止的机制和调控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被用来详细描述沿着基本转录循环途径的构象转变和中间产物的序列、大小、动力学和顺序。我们将研究原核转录机器亚单位和真核转录机器亚单位的瞬时蛋白质-DNA和蛋白质-蛋白质相互作用(及其诱导的构象)。还将以几种转录因子作为模型系统来探讨原核和真核转录调控的机制。在自由扩散的转录复合体和固定化的复合体上实施的几种单分子分析形式将分别用于解决机理和动力学问题。这些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控中的作用，为这些研究开发的工具可以推广到调节电路和其他生物分子机制的研究。原核转录的研究将为理解抗菌药物(通过抑制构象)提供基础，从而为药物设计和筛选提供相关的洞察力和分析方法。真核转录的研究将为理解转录相关疾病的机制奠定基础，并将有助于设计和筛选治疗这些疾病的药物。与公共健康相关：拟议的工作将揭开基因表达的第一步也是最重要的一步，即转录和转录调控的详细机制。这将为详细了解抗菌剂的分子和结构作用铺平道路。它还将为几个长期存在的和未解决的问题提供答案，这些问题涉及到真核转录的早期启动和调控。后者将提供对转录相关疾病病因的详细分子理解，并将为预防、管理和治愈这些疾病的药物设计和筛选提供工具。

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.