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: 8171532
• 负责人: JEFFREY W ROBERTS
• 金额: $ 0.89万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171532
• 关键词: Archaea; Bacteria; Base Sequence; Binding; Biochemical; Biochemical Genetics; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA-Directed RNA Polymerase; Elongation Factor; Enzymes; Escherichia coli; Funding; Genetic Transcription; Grant; Human; Institution; Ions; Mediating; Modeling; Mycobacterium tuberculosis; Play; Polymerase; RNA Polymerase II; RNA chemical synthesis; Regulation; Research; Research Personnel; Resources; Role; Saccharomyces cerevisiae; Solutions; Source; Staging; Structure; System; Thermus; Thermus thermophilus; Transcription Elongation; Transcriptional Regulation; United States National Institutes of Health; Zinc; antitermination; insight; transcription termination

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 大肠杆菌RNA聚合酶 - 苏氏菌的晶体结构和抗终止机理。 RNA（转录）的合成是由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伸长和终止阶段。 NUSA是一种重要的多功能转录伸长因子NUSA。根据核苷酸序列以及其他因素的存在或不存在，NUSA（55 kDa）靶向RNAP核心酶（〜400-KDA）以刺激转录暂停或抑制转录暂停和介导抗验证。由于缺乏RNAP-NUSA复杂结构，其显然对转录伸长和终止的影响尚未完全理解。 为了深入了解转录伸长和终止中的NUSA功能，我们与NUSA进行了共结合的RNAP，均从大肠杆菌（E.Coli）纯化。即使大肠杆菌RNAP是一种特征良好的多生物酶，广泛用于研究转录调节的生化系统中，但尚未确定其结构。因此，在热水生植物和热嗜热RNAP的晶体结构中重新检查了在大肠杆菌中进行的转录调节的生化和遗传研究。它们的一部分结构在大细菌和古细菌之间是强烈的保守，但是许多地区，尤其是潜在参与调节的区域，与大肠杆菌RNAP不同。同样，除了结核分枝杆菌的截短的NUSA晶体结构外，没有其他大肠杆菌NUSA的结构。 我们建议使用来自RNAP中本质上固定的锌离子的单个异常衍射（SAD）来确定大肠杆菌RNAP和E.COLI NUSA之间识别复合物的共晶结构。最近，这种悲伤的方法允许酿酒酵母RNA聚合酶II（POLII）模型的12个亚基糖酵母模型完全完善至3.8a。在这一点上，当我们新获得共同体时，我们还不知道共晶的衍射质量。我们最近获得了RNAP-NUSA的共晶，并建议使用MacChess设施来确定其衍射质量作为迈向结构解决方案的第一步。