X-Ray Crystallographic Studies of N4 RNA Polymerases

描述(申请人提供)：转录是基因表达的主要控制点，而RNA聚合酶(RNAP)是这一过程中调控的中心靶标。我们的长期目标是了解RNAP及其调控的机制和功能。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、结晶并解决了噬菌体N4编码的病毒粒子包裹的RNAP(总共3,500个氨基酸残基)功能性RNAP结构域(1106个氨基酸残基，mini-vRNAP)的2.0 Angstroms分辨率的X射线晶体结构。这是我们工作的重大突破。在这里，我建议进行进一步的结构研究，以增加我们对RNAP功能和转录机制的了解。具体目标是：1.解决mini-vRNAP与启动子DNA二元复合体的晶体结构。我已经得到了有希望的二元络合物的晶体；2.对转录起始过程进行了结构表征。找到了制备转录起始复合体(活性部位含有两个底物核苷酸的二元复合体)的条件；3.解决了转录延伸复合体的晶体结构。我们将制备迷你vRNAP转录延伸复合物用于结晶，并解决结构问题。4.解析N4 RNAPII的晶体结构。我们将结晶并解决N4 RNAPII和RNAPII与GP2的复合体的结构，GP2是一种重要的辅因子。这些研究将为T7噬菌体样单亚基RNAP家族的转录机制提供见解，特别是该家族中需要辅助因子的那些酶。

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.