CRYSTAL STRUCTURE OF ECOLI RNA POLYMERASE-NUSA AND MECHANISM OF

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 大肠杆菌RNA聚合酶NusA的晶体结构及其抗终止机制。 RNA的合成(转录)是由DNA依赖的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。许多因素通过直接与RNAP的特定区域结合来影响转录的延伸和终止阶段。在细菌的转录终止和反终止中发挥如此重要作用的一个有趣的因素是NusA，一种重要的多功能转录延伸因子。根据核苷酸序列和其他因子的存在与否，NusA(55-kDa)以RNAP核心酶(~400-kDa)为靶标，刺激转录暂停，或抑制转录暂停，介导抗终止。由于缺乏RNAP-NusA复合体结构，它对转录延伸和终止的明显相反作用尚未完全清楚。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，这两种RNAP都是从大肠杆菌中纯化的。尽管E.coliRNAP是一种特性良好的多亚单位酶，广泛用于高度精炼的生化系统来研究转录调控，但其结构尚未确定。因此，在E.Coli中进行的转录调控的生化和遗传学研究已经在Thermus Aquaticus和Thermus thermophilus RNAP的晶体结构中重新进行了检查。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控的区域，与E.ColiRNAP的结构不同。同样，除了来自结核分枝杆菌的截短的NusA晶体结构外，没有可用的E.coliNusA结构。我们建议利用RNAP中固有结合的锌离子的单次反常衍射(SAD)来确定E.coliRNAP和E.coliNusA之间识别络合物的共晶结构。最近，SAD方法使酿酒酵母RNA聚合酶II(POL II)模型完全精炼到3.8A。在这一点上，我们还不知道我们的共晶的衍射质量，因为我们新获得了共晶。我们最近获得了RNAP-NUSA的共晶，并建议使用MacCHESS设备来确定它们的衍射质量，作为迈向结构解决方案的第一步。

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.