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：
8326636
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 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8326636
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）：该提案试图在原核转录启动，伸长和终止以及真核转录开始的早期阶段的机制和调节中阐明未解决的问题。单分子荧光光谱和显微镜方法将用于详细介绍沿基底转录周期途径的构象转变和中间体的序列，大小，动力学和顺序。瞬时蛋白-DNA和蛋白质 - 蛋白质相互作用（及其诱导的构象）将研究核酸转录机器的亚基和一些真核转录机械的亚基。还将使用几种转录因子作为模型系统探索原核生物和真核转录调节的机制。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格式将分别用于解决机械和动力学问题。拟议的研究预计将阐明构象转变和转录中的生物分子相互作用的作用，并且为这些研究开发的工具可以推广用于研究调节回路和其他生物分子机械的研究。拟议的核转录研究将为理解抗菌药物（通过抑制构象）提供基础，因此将为药物设计和筛查提供相关的见解和测定。对真核转录的拟议研究将为理解与转录相关的人类疾病的机制奠定基础，并有助于为这些疾病设计和筛查治疗剂。公共卫生相关性：拟议的工作将揭示基因表达中第一个也是最重要的步骤的详细机制，即转录和转录调节。它将为对抗菌剂作用的详细分子和结构理解铺平道路。它还将为关于真核转录启动和调节的早期阶段的几个长期尚未解决的问题提供答案。后者将提供对转录相关疾病的原因的详细分子理解，并将为药物设计和筛查提供工具，以预防，管理和治愈这些疾病。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Parallel multispot smFRET analysis using an 8-pixel SPAD array.

DOI：
10.1117/12.909470
发表时间：
2012-01-21
期刊：
Proceedings of SPIE--the International Society for Optical Engineering
影响因子：
0
作者：
Ingargiola A;Colyer RA;Kim D;Panzeri F;Lin R;Gulinatti A;Rech I;Ghioni M;Weiss S;Michalet X
通讯作者：
Michalet X

Different types of pausing modes during transcription initiation.

转录起始期间不同类型的暂停模式。

DOI：
10.1080/21541264.2017.1308853
发表时间：
2017
期刊：
Transcription
影响因子：
0
作者：
Lerner,Eitan;Ingargiola,Antonino;Lee,JookyungJ;Borukhov,Sergei;Michalet,Xavier;Weiss,Shimon
通讯作者：
Weiss,Shimon

Probing biomolecular structures and dynamics of single molecules using in-gel alternating-laser excitation.