X-Ray Crystallographic Studies of N4 RNA Polymerases

N4 RNA 聚合酶的 X 射线晶体学研究

基本信息

批准号：
7469525
负责人：
Katsuhiko Murakami
金额：
$ 21.06万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2010-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): 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 Angstroms 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 characterize the transcription initiation process. 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.

描述（由申请人提供）：转录是基因表达和RNA聚合酶（RNAP）的主要控制点，是此过程中调节的核心目标。我们的长期目标是了解RNAP及其调节的机制和功能。除了生化和生物物理方法外，确定高分辨率的三维结构是必不可少的一步。我最近纯化，结晶和解决了噬菌体N4编码的毒piRion包含的RNAP的功能性RNAP结构域（1106个氨基酸残基，微量VRNAP）的2.0埃轴分辨率X射线晶体结构（总计3,500氨基酸残基）。这代表了我们工作的重大突破。在这里，我提出了进一步的结构研究，旨在提高我们对RNAP功能和转录机理的理解。具体目的是：1。求解小型VRNAP和启动子DNA二进制复合物的晶体结构。我获得了二元复合物的有希望的晶体。 2。在结构上表征转录启动过程。我发现了准备转录起始复合物（活性位点中有两个底物核苷酸的二元复合物）的条件； 3。求解转录伸长复合物的晶体结构。我们将准备小型VRNAP转录伸长复合物，以结晶并解决结构。 4。解决N4 RNAPII的晶体结构。我们将用GP2（一种必需的辅助因子GP2）结晶并解决N4 RNAPII和RNAPII复合物的结构。这些研究将提供有关T7噬菌体样单人物RNAP家族转录机制的见解，尤其是家族中需要辅助因子的那些酶。