X-Ray Crystallographic Studies of N4 RNA Polymerases

描述（由申请人提供）：转录是基因表达的主要控制点，RNA聚合酶（RNAP）是这一过程的中心调控靶点。我们的长期目标是了解RNAP的机制和功能及其调控。除了生物化学和生物物理方法，确定高分辨率的三维结构是必不可少的一步。我最近对噬菌体n4编码的病毒粒子包封的RNAP（共3500个氨基酸残基）的功能性RNAP结构域（1106个氨基酸残基，mini-vRNAP）的2.0埃分辨率x射线晶体结构进行了纯化、结晶和求解。这是我们工作中的一个重大突破。在这里，我建议进一步的结构研究，旨在增加我们对RNAP功能和转录机制的理解。具体目标是：1.；求解微型vrnap和启动子DNA二元复合物的晶体结构。我已经得到了有希望的二元配合物晶体；2. 转录起始过程的结构特征。我已经找到了准备转录起始复合物的条件（活性位点有两个底物核苷酸的二元复合物）；3. 求解一个转录延伸复合物的晶体结构。我们将制备mini-vRNAP转录延伸配合物进行结晶并求解其结构。4. 求解N4 RNAPII的晶体结构。我们将结晶和解决N4 RNAPII和RNAPII复合物的结构与gp2，一个重要的辅因子。这些研究将有助于深入了解T7噬菌体样单亚基RNAP家族的转录机制，特别是该家族中需要辅助因子的酶。

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.