Molecular mechanisms of transcription elongation

转录延伸的分子机制

基本信息

批准号：
7898188
负责人：
DMITRY G VASSYLYEV
金额：
$ 14.7万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-13 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7898188
关键词：
Antibiotics Arts Bacteria Bacterial Model Bacterial RNA Bacteriophages Binding Biochemical Biochemical Genetics Biological Models Catalysis Classification Complex Crystallization DNA DNA-Directed RNA Polymerase Data Development Diphosphates Elements Elongation Factor Enzyme Gene Enzymes Escherichia coli Eukaryota Gene Expression Genetic Genetic Transcription Goals Human Lateral Lead Life Malignant Neoplasms Messenger RNA Modeling Molecular Molecular Conformation Molecular Models Molecular Weight Movement Nucleic Acids Nucleotides Organism Pattern Phase Play Proteins RNA Regulator Genes Research Personnel Resolution Resources Roentgen Rays Role Signal Transduction Site Site-Directed Mutagenesis Small RNA Solutions Source Structure System T7 RNA polymerase Testing Thermodynamics Thermus thermophilus Transcript Transcription Elongation Transcriptional Regulation Variant Virulence Work Yeasts analog base conformational conversion design ds-DNA in vivo infancy insight molecular modeling neoplastic novel nucleotide analog programs promoter research study scaffold single molecule

项目摘要

RNA polymerase is the central enzyme of gene expression in all domains of life. Changes in gene expression patterns punctuate the progress of normal and neoplastic development of eukaryotes and onset of virulence in bacteria. Elongation is the longest and a highly regulated part of transcription cycle. Elongation factors such as PTEF-b and RfaH are implicated in development of cancer in humans and virulence in bacteria, respectively. This project aims at elucidation of the detailed molecular mechanism of transcript elongation by bacterial RNA polymerase by combining the high-resolution structural analysis of elongation intermediates with the state-of-the-art biochemical characterization of the elongation mechanism. Different states of the transcription complex that have been detected by biochemical and single-molecule experiments likely serve as targets for protein factors, small RNAs, nucleotide analogs, antibiotics, and other regulators of gene expression. Structures of RNA polymerase complexes that represent functional intermediates in the transcription cycle will be determined for the two model bacterial systems, Escherichia coli and Thermus thermophilus. The former is the major source of the biochemical and genetic data, whereas the latter afforded the high-resolution RNAP structures. Specific aims of this study will be as follows. First, to determine organization and structure of the transcription elongation complex at the atomic level. Nucleic acid scaffolds will be used to assemble, characterize, and crystallize functional elongation complexes captured in different conformational states and to solve their structures. Structure-based site-directed mutagenesis will be then applied to examine functional importance of the key elements of the transcription elongation complex. Second, to elucidate the mechanism of substrate selection and catalysis by bacterial RNA polymerase, structures of the transcription elongation complexes with the incoming substrates will be determined by X-ray analysis. Subsequent functional tests will be designed to identify the determinants for nucleotide selection and incorporation; in parallel, genetic approaches will be used to study the regulation of transcriptional fidelity in vivo. Third, to elucidate the elongation complex determinants that control both its high thermodynamic stability and the facile translocation upon nucleotide addition, the effects of systematic variation of the scaffold components and substitutions of the key RNA polymerase residues that are highlighted by the structure will be analyzed

RNA聚合酶是生命所有领域中基因表达的中心酶。基因表达模式的变化突出了真核生物正常和肿瘤发育的进展以及细菌中毒力的发作。伸长是转录周期中最长和高度调节的一部分。 PTEF-B和RFAH等延伸因子分别涉及人类癌症的发展和细菌中的毒力。该项目旨在通过将伸长中间体的高分辨率结构分析与伸长机制的最新生化特征相结合，从而阐明细菌RNA聚合酶的详细分子机制。通过生化和单分子实验检测到的转录复合物的不同状态可能是蛋白质因子，小RNA，核苷酸类似物，抗生素和其他基因表达调节剂的靶标。代表转录周期中功能性中间体的RNA聚合酶复合物的结构将确定针对两个模型的细菌系统Escherichia Coli和Thermus Thermophilus。前者是生化和遗传数据的主要来源，而后者则提供了高分辨率的RNAP结构。这项研究的具体目标将如下。首先，确定在原子水平上转录伸长复合物的组织和结构。核酸支架将用于组装，表征和结晶的功能伸长络合物，这些功能伸长络合物在不同的构象状态下捕获并解决其结构。然后，将基于结构的位置定向诱变来检查转录伸长复合物的关键要素的功能重要性。其次，为了阐明细菌RNA聚合酶的底物选择和催化的机理，将通过X射线分析确定转录伸长复合物与传入底物的结构。随后的功能测试将旨在识别核苷酸选择和掺入的决定因素。同时，遗传方法将用于研究体内转录忠诚度。第三，为了阐明控制其高热力学稳定性和添加核苷酸添加时的便捷易位的延伸复合物决定因素，将分析由脚手架成分的系统变化的效果以及关键RNA聚合酶残基的替代，这些酶是由结构突出显示的

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Conformational transition of Sec machinery inferred from bacterial SecYE structures.

DOI：
10.1038/nature07421
发表时间：
2008-10-16
期刊：
NATURE
影响因子：
64.8
作者：
Tsukazaki, Tomoya;Mori, Hiroyuki;Fukai, Shuya;Ishitani, Ryuichiro;Mori, Takaharu;Dohmae, Naoshi;Perederina, Anna;Sugita, Yuji;Vassylyev, Dmitry G.;Ito, Koreaki;Nureki, Osamu
通讯作者：
Nureki, Osamu

Elongation by RNA polymerase: a race through roadblocks.

RNA 聚合酶的延伸：一场跨越障碍的竞赛。