X-Ray Crystallographic Studies of N4 RNA Polymerases

N4 RNA 聚合酶的 X 射线晶体学研究

• 批准号: 7472814
• 负责人: Katsuhiko Murakami
• 金额: $ 2.18万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472814
• 关键词: Active Sites; Amino Acids; Bacteriophage N4; Bacteriophage T7; Bacteriophages; Binding; Biochemical; Code; Complex; Condition; Crystallization; DNA; DNA Structure; DNA-Directed RNA Polymerase; Dissociation; Encapsulated; Enzymes; Escherichia coli; Family; Functional RNA; Gene Expression; Genetic Transcription; Goals; Individual; Length; Light; Nucleotides; Object Attachment; Process; RNA Sequences; Range; Recruitment Activity; Regulation; Research Personnel; Resolution; Roentgen Rays; Role; SS DNA BP; Single-Stranded DNA; Staging; Structure; Structure-Activity Relationship; System; T7 RNA polymerase; Transcript; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Activation; Virion; Work; cofactor; in vivo; insight; phosphodiester; programs; promoter; protein protein interaction; scaffold; three dimensional structure; tripolyphosphate

Transcription is the major control point of gene expression and RNA polymerase (RNAP) is the central target of regulation in this process. Our long term goal is to understand the mechanisms and functions of RNAP and its regulation. In addition to biochemical and biophysical approaches, determining high resolution three- dimensional structures is an essential step. I recently purified, crystallized, and solved the 2.0 _ resolution X- ray crystal structure of the functional RNAP domain (1106 amino acid residues, mini-vRNAP) of bacteriophage N4-encoded virion-encapsulated RNAP (total 3,500 amino acid residues). This represents a major breakthrough in our work. Here, I propose further structural studies aimed towards increasing our understanding of RNAP function and the mechanism of transcription. Specific aims are: 1. Solve crystal structure of mini-vRNAP and promoter DNA binary complex. I have obtained promising crystals of the binary complex; 2. Structurally eharaeterize the transeription initiation proeess. I have found conditions for preparing a transcription initiation complexes (binary complex with two substrate nucleotides in active site); 3. Solve crystal structure of a transcription elongation complex. We will prepare mini-vRNAP transcription elongation complexes for crystallization and solve the structure. 4. Solve the crystal structure of N4 RNAPII. We will crystallize and solve the structures of N4 RNAPII and RNAPII complex with gp2, an essential cofactor. These studies will provide insights into the mechanism of transcription of the T7 bacteriophage-like single-subunit RNAP family, and specifically those enzymes in the family that require accessory factors. I I

转录是基因表达的主要控制点，RNA聚合酶（RNAP）是中心靶标 这个过程中的监管。我们的长期目标是了解RNAP和RNAP的机制和功能 其监管。除了生物化学和生物物理方法外，确定高分辨率三 维度结构是必不可少的一步。我最近提纯、结晶、解出了2.0_分辨率X- 功能性 RNAP 结构域（1106 个氨基酸残基，mini-vRNAP）的射线晶体结构 噬菌体 N4 编码的病毒粒子封装的 RNAP（总共 3,500 个氨基酸残基）。这代表了一个 我们的工作取得重大突破。在这里，我建议进一步的结构研究，旨在提高我们的 了解 RNAP 功能和转录机制。具体目标是： 1. 解决晶体 mini-vRNAP 和启动子 DNA 二元复合物的结构。我已经获得了有前途的晶体 二元复合体； 2. 从结构上描述转录启动过程。我已找到条件 用于制备转录起始复合物（具有活性的两个底物核苷酸的二元复合物） 地点）; 3. 解析转录延伸复合物的晶体结构。我们将准备mini-vRNAP 转录延伸复合物用于结晶并解决结构。 4. 解决晶体结构 N4 RNAPII。我们将结晶并解析 N4 RNAPII 和 RNAPII 与 gp2 的复合物的结构， 必需的辅助因子。这些研究将深入了解 T7 的转录机制 噬菌体样单亚基 RNAP 家族，特别是家族中需要的那些酶 附属因素。 我 我