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

项目摘要该建议介绍了细菌RNA聚合酶的转录启动，伸长和终止（RNAP）。在转录启动时，RNAP：（i）与启动子DNA结合，得出RNAP促进膜闭合复合物；（ii）放开启动子DNA，产生RNAP启动器开放式复合物；（iii）合成第一个〜11个核苷酸 RNA作为RNAP启动器初始转录复合物，使用RNAP的“屈服”机制保持启动子DNA的静止，并在每个核苷酸 - 增添循环中拉动相邻的DNA；（iv）从发起人逃脱。在转录伸长率中，RNAP合成RNA的其余核苷酸作为RNAP-DNA伸长复合物，使用“踏脚”机制，其中RNAP在DNA上向前移动在每个核苷酸 - 结核周期中。在转录终止中，RNAP停止合成RNA，释放RNA，并与DNA分离。这些反应中的每一个都是调节剂的目标。了解转录启动，伸长，终止和调节将需要定义蛋白质和每种反应中的DNA，结构过渡的动力学以及调节器影响的机制结构过渡。在当前时期，我们定义了转录起始站点选择的结构基础，从头转录启动，非典型发射核苷酸依赖性转录启动，初始转录和II类转录激活；我们开发了高通量测序方法，使得能够综合分析转录的DNA序列决定因素；我们开发了多路复用的交联方法这可以全面分析转录中的蛋白-DNA相互作用；我们开发了合奏，单分子荧光测定，可以监测RNAP夹和RNAP触发环形环溶液中的构象；我们定义了与小分子抑制剂的结合位点和机制转录。拟议的工作将基于当前时期的发现。拟议的工作将使用X射线晶体学，单分子生物物理学，生物化学和遗传学，以解决五个具体目标：特定目的1：确定RNAP打滑的结构基础特定目的2：确定RNAP易位的结构基础特定目的3：分析RNAP伸长，暂停和终止中的RNAP易位特定目的4：分析RNAP夹具构象中的伸长，暂停和终止特定目的5：分析RNAP触发环构象在伸长，暂停和终止中