CRYSTALLOGRAPHIC STUDIES OF DNA AND RNA POLYMERASES

DNA 和 RNA 聚合酶的晶体学研究

• 批准号: 8361608
• 负责人: THOMAS Arthur STEITZ
• 金额: $ 4.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361608
• 关键词: Binding; Biochemical; Complex; Cyclic AMP Receptor Protein; DNA; DNA Polymerase III; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; DnaB helicase; DnaG Protein; Escherichia coli; Funding; Goals; Grant; Lac Operon; National Center for Research Resources; Nucleotides; Phase; Polymerase; Principal Investigator; Protein Binding; Proteins; RNA; Regulation; Research; Research Infrastructure; Resources; Series; Source; Structure; Thermus; Transcription Initiation; United States National Institutes of Health; base; cost; helicase; promoter; research study; structural biology; tau Proteins; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to provide a structural basis for understanding the mechanisms of initiation and elongation by DNA and RNA polymerases, their transitions from initiation to elongation phases, the regulation of polymerases by factors and the mechanisms by which these polymerases assure that the correct nucleotide is inserted. This objective will be achieved by determining the crystal structures of polymerases complexed with functionally associated proteins and bound to appropriate DNA or RNA substrates, as well as by appropriate biochemical experiments. Further, we aim to establish the structures of component assemblies of the replisome. These include a replication fork complex between DNA polymerase III, tau and a forked-DNA substrate, as well as the structure of the primasome that includes Thermus aquaticus DnaB helicase complexed with the DnaG primase bound to their DNA substrate. Additionally, the structure of the DnaB helicase bound to its DNA and nucleotide substrates as well as the helicase binding domain of DnaG and helicase loader protein. The structural basis of the initiation of DNA synthesis by the protein primed phi29 DNA polymerase and the mechanism of its transition to the elongation phase will be determined through a series of structures of intermediate states in this transition. The regulation of transcription initiation by the multi-subunit RNA polymerase from E. coli will be examined from its structures complexed with the gal and lac operon promoter DNAs and the catabolite gene activator protein regulator transcription factor.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 该项目的总体目标是为理解DNA和RNA聚合酶的起始和延伸机制、它们从起始阶段到延伸阶段的转变、聚合酶受因子的调节以及这些聚合酶确保插入正确核苷酸的机制提供结构基础。这一目标将通过确定与功能相关蛋白质复合并与适当的DNA或RNA底物结合的聚合酶的晶体结构以及通过适当的生物化学实验来实现。此外，我们的目标是建立复制体的组件组装的结构。这些包括DNA聚合酶III、tau和叉状DNA底物之间的复制叉复合物，以及包括与结合到其DNA底物的DnaG引发酶复合的水生栖热菌DnaB解旋酶的引发体的结构。此外，DnaB解旋酶的结构结合到其DNA和核苷酸底物以及DnaG和解旋酶装载蛋白的解旋酶结合结构域。通过蛋白质引发的phi 29 DNA聚合酶启动DNA合成的结构基础及其向延伸阶段过渡的机制将通过该过渡中的一系列中间状态的结构来确定。研究了大肠杆菌多亚基RNA聚合酶对转录起始的调控作用。将从其与gal和lac操纵子启动子DNA和分解代谢物基因激活蛋白调节转录因子复合的结构来检查大肠杆菌。