CRYSTAL STRUCTURE OF ECOLI RNA POLYMERASE-NUSA AND MECHANISM OF

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。一个有趣的因素发挥这样的整体作用，在转录终止和反终止的细菌是NusA，一个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）靶向RNAP核心酶（~400-kDa）以刺激转录暂停，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作用尚未完全理解。 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是一种广泛用于高度精细生化系统以研究转录调控的充分表征的多亚基酶，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物化学和遗传学研究已经在水生栖热菌和嗜热栖热菌RNAP的晶体结构中重新检查。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，与大肠杆菌RNAP不同。类似地，除了来自结核分枝杆菌的截短的NusA晶体结构之外，没有可用的大肠杆菌NusA结构。 我们建议使用单个异常衍射（SAD）从固有地结合在RNAP中的锌离子确定大肠杆菌RNAP和大肠杆菌NusA之间的识别复合物的共晶结构。最近SAD方法允许12-亚基酿酒酵母RNA聚合酶II（Pol II）模型被完全细化至3.8A。在这一点上，我们还不知道我们的共晶衍射质量，因为我们新获得的共晶。我们最近收购了RNAP-NusA的共晶，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。

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.