CRYSTAL STRUCTURE OF ECOLI RNA POLYMERASE-NUSA AND MECHANISM OF

大肠杆菌RNA聚合酶-NUSA的晶体结构及其作用机制

• 批准号: 8363540
• 负责人: JEFFREY W ROBERTS
• 金额: $ 2.38万
• 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: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363540
• 关键词: Archaea; Bacteria; Base Sequence; Binding; Biochemical; Biochemical Genetics; Complex; DNA-Directed RNA Polymerase; Elongation Factor; Enzymes; Escherichia coli; Funding; Genetic Transcription; Grant; Human; Ions; Mediating; Modeling; Mycobacterium tuberculosis; National Center for Research Resources; Play; Principal Investigator; RNA Polymerase II; RNA chemical synthesis; Regulation; Research; Research Infrastructure; Resources; Role; Saccharomyces cerevisiae; Solutions; Source; Staging; Structure; System; Thermus; Thermus thermophilus; Transcription Elongation; Transcriptional Regulation; United States National Institutes of Health; Zinc; antitermination; cost; insight; transcription termination

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. Crystal structure of E.coli RNA polymerase-NusA and mechanism of anti-termination. The synthesis of RNA (transcription) is mediated by DNA-dependent RNA polymerases (RNAP) whose structure and function are highly conserved from bacteria to human. A number of factors influence the elongation and termination stages of transcription by directly binding to specific regions of RNAP. One intriguing factor playing such an integral role in transcription termination and anti-termination in bacteria is NusA, an essential multifunctional transcription elongation factor. Depending on the nucleotide sequence and the presence or absence of other factors, NusA (55-kDa) targets RNAP core enzyme (~400-kDa) to stimulate transcription pausing, or inhibit transcription pausing and mediate antitermination. Its apparently opposing effects on transcription elongation and termination have not been fully understood because of the lack of the RNAP-NusA complex structure. To gain insight into NusA function in transcription elongation and termination, we have cocrystallized RNAP with NusA, both purified from Escherichia coli (E.coli). Even though E.coli RNAP is a well characterized multi-subunit enzyme widely used in highly refined biochemical systems to study transcription regulation, its structure has not been determined. Hence, biochemical and genetic studies of transcription regulation done in E.coli have been re-examined in the crystal structures of Thermus aquaticus and Thermus thermophilus RNAP. Parts of their structures are strongly conserved among eubacterial and archaea, but many regions, particularly those potentially involved in regulation, are different from that of E.coli RNAP. Similarly, there is no structure of E.coli NusA available other than truncated NusA crystal structures from Mycobacterium tuberculosis. We propose to determine a cocrystal structure of the recognition complex between E.coli RNAP and E.coli NusA using the single anomalous diffraction (SAD) from zinc ions bound intrinsically in RNAP. Recently the SAD approach allowed the 12-subunit Saccharomyces cerevisiae RNA polymerase II (Pol II) model to be fully refined up to 3.8A. At this point, we do not know the diffraction quality of our cocrystals yet as we newly acquired the cocrystals. We recently acquired the cocrystals of RNAP-NusA and propose to use MacCHESS facility to determine their diffraction quality as the first step towards the structure solution.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 大肠杆菌RNA聚合酶-NusA的晶体结构及抗终止机制。 RNA 的合成（转录）是由 DNA 依赖性 RNA 聚合酶 (RNAP) 介导的，其结构和功能从细菌到人类都高度保守。许多因素通过直接结合 RNAP 的特定区域来影响转录的延伸和终止阶段。 NusA 是一种在细菌转录终止和反终止中发挥重要作用的有趣因子，它是一种重要的多功能转录延伸因子。根据核苷酸序列以及是否存在其他因素，NusA (55-kDa) 以 RNAP 核心酶 (~400-kDa) 为目标，刺激转录暂停，或抑制转录暂停并介导抗终止。由于缺乏 RNAP-NusA 复合物结构，其对转录延伸和终止的明显相反的作用尚未完全了解。 为了深入了解 NusA 在转录延伸和终止中的功能，我们将 RNAP 与 NusA 共结晶，两者均从大肠杆菌 (E.coli) 中纯化。尽管大肠杆菌 RNAP 是一种特征明确的多亚基酶，广泛用于高度精细的生化系统中以研究转录调控，但其结构尚未确定。因此，在大肠杆菌中进行的转录调控的生化和遗传学研究已在水生栖热菌和嗜热栖热菌 RNAP 的晶体结构中得到了重新检验。它们的部分结构在真细菌和古细菌中高度保守，但许多区域，特别是那些可能参与调节的区域，与大肠杆菌 RNAP 的不同。同样，除了结核分枝杆菌的截短 NusA 晶体结构外，没有可用的大肠杆菌 NusA 结构。 我们建议使用 RNAP 中固有结合的锌离子的单反常衍射 (SAD) 来确定大肠杆菌 RNAP 和大肠杆菌 NusA 之间的识别复合物的共晶结构。最近，SAD 方法使 12 亚基酿酒酵母 RNA 聚合酶 II (Pol II) 模型完全细化至 3.8A。此时，我们还不知道共晶体的衍射质量，因为我们是新获得的共晶体。我们最近获得了 RNAP-NusA 的共晶体，并建议使用 MacCHESS 设备来确定其衍射质量，作为结构解决方案的第一步。