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Bacterial Transcription Complexes

细菌转录复合物

基本信息

批准号：
10388566
负责人：
RICHARD H. EBRIGHT
金额：
$ 5.1万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2022-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10388566
关键词：
Address Affect Anti-Bacterial Agents Bacterial Genes Bacterial RNA Binding Binding Sites Biochemistry Biological Assay Biophysics Closure by clamp Complex DNA DNA Sequence Analysis DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase DNA-Protein Interaction Electron Transport Complex III Fluorescence Gene Expression Genetic Genetic Transcription Growth High-Throughput Nucleotide Sequencing Kinetics Molecular Conformation Molecular Machines Monitor Nucleotides Pharmaceutical Preparations Protein Analysis Proteins RNA RNA Polymerase I RNA analysis Reaction Regulation Structure Transcription Elongation Transcription Initiation Transcription Initiation Site Transcriptional Activation Work X-Ray Crystallography crosslink design improved novel promoter single molecule small molecule inhibitor transcription termination

项目摘要

Project Summary This proposal addresses transcription initiation, elongation, and termination by bacterial RNA polymerase (RNAP). In transcription initiation, RNAP: (i) binds to promoter DNA, yielding an RNAP-promoter closed complex; (ii) unwinds promoter DNA, yielding an RNAP-promoter open complex; (iii) synthesizes the first ~11 nucleotides of RNA as an RNAP-promoter initial transcribing complex, using a "scrunching" mechanism in which RNAP remains stationary on promoter DNA and pulls in adjacent DNA in each nucleotide-addition cycle; and (iv) escapes from the promoter. In transcription elongation, RNAP synthesizes the remaining nucleotides of RNA as an RNAP-DNA elongation complex, using a "stepping" mechanism in which RNAP moves forward on DNA in each nucleotide-addition cycle. In transcription termination, RNAP stops synthesizing RNA, releases RNA, and dissociates from DNA. Each of these reactions is a target for regulators. Understanding transcription initiation, elongation, termination, and regulation will require defining the structural transitions in protein and DNA in each reaction, the kinetics of structural transitions, and the mechanisms by which regulators affect structural transitions. In the current period, we defined the structural basis of transcription start-site selection, de novo transcription initiation, non-canonical-initiating-nucleotide-dependent transcription initiation, initial transcription, and Class II transcription activation; we developed high-throughput-sequencing approaches that enable comprehensive analysis of DNA-sequence determinants for transcription; we developed multiplexed crosslinking approaches that enable comprehensive analysis of protein-DNA interactions in transcription; we developed ensemble and single-molecule fluorescence assays that enable monitoring of RNAP clamp and RNAP trigger-loop conformation in solution; and we defined binding sites and mechanisms for small-molecule inhibitors of transcription. The proposed work will build on the findings of the current period. The proposed work will use x-ray crystallography, single-molecule biophysics, biochemistry, and genetics to address five specific aims: Specific Aim 1: Determination of the structural basis of RNAP slippage Specific Aim 2: Determination of the structural basis of RNAP translocation Specific Aim 3: Analysis of RNAP translocation in elongation, pausing, and termination Specific Aim 4: Analysis of RNAP clamp conformation in elongation, pausing, and termination Specific Aim 5: Analysis of RNAP trigger-loop conformation in elongation, pausing, and termination

项目总结