X-Ray Crystallographic Studies of N4 RNA Polymerases

转录是基因表达的主要控制点，而RNA聚合酶(RNAP)是基因表达的中心靶点 在这一过程中的监管。我们的长期目标是了解RNAP的机制和功能，以及 它的规定。除了生化和生物物理方法外，测定高分辨率的三个- 维度结构是必不可少的一步。我最近提纯、结晶并解决了2.0分辨率的X- 中华乳杆菌功能性RNAP结构域(1106个氨基酸残基，mini-vRNAP)的射线晶体结构 噬菌体N4编码病毒粒子包裹的RNAP(共3,500个氨基酸残基)。这表示一个 --工作取得重大突破。在这里，我建议进行进一步的结构研究，旨在增加我们的 了解RNAP的功能和转录机制。具体目标是：1.解决水晶 Mini-vRNAP与启动子DNA二元复合体的结构。我已经得到了很有希望的晶体 二元复合体；2.从结构上优化转导起始过程。我已经找到了条件 用于制备转录起始复合体(具有活性的两个底物核苷酸的二元复合体 3.解决转录延伸复合体的晶体结构。我们将准备mini-vRNAP 转录延伸复合体进行结晶并解决结构问题。4.解决晶体结构问题 N4RNAPII。我们将结晶和解决N4 RNAPII和RNAPII与GP2，和 基本辅因子。这些研究将提供对T7转录机制的深入了解 类噬菌体单亚单位RNAP家族，特别是家族中需要 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