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.

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