X-Ray Crystallographic Studies of N4 RNA Polymerases

转录是基因表达的主要控制点，RNA聚合酶（RNAP）是其中心靶点 在这个过程中的监管。我们的长期目标是了解RNAP的机制和功能， 其规定。除了生物化学和生物物理方法，确定高分辨率的三个- 三维结构是必不可少的一步。我最近纯化，结晶，并解决了2.0 _分辨率X- 的功能RNAP结构域（1106个氨基酸残基，mini-vRNAP）的射线晶体结构。 噬菌体N4编码的病毒体包封的RNAP（总共3，500个氨基酸残基）。这表示 我们工作的重大突破。在这里，我建议进一步的结构研究，旨在增加我们的 了解RNAP的功能和转录机制。具体目标是：1。解晶 mini-vRNAP和启动子DNA二元复合物结构。我得到了很有希望的 二元复合体; 2.从结构上描述了传递起始过程。我发现有条件 用于制备转录起始复合物（具有两个活性底物核苷酸的二元复合物 网站）; 3.解出转录延伸复合物的晶体结构。我们将准备迷你vRNAP 转录延伸复合物的结晶和解决结构。4.解晶体结构 N4 RNAPII我们将结晶并解析N4 RNAPII和RNAPII与gp 2的复合物的结构， 基本辅因子这些研究将为深入了解T7基因的转录机制提供帮助。 噬菌体样单亚基RNAP家族，特别是家族中那些需要 辅助因素 我 我

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