Structural-Function Studies of Shared hRNAP Components

描述（由申请人提供）：本项目的长期目标是 了解gent基因的转录调控机制， 在分子水平上表达。所有真核细胞核的转录 基因由三种不同的RNI聚合酶（RP）I、II和III进行， 分别合成核糖体、信使和转运RNA。RP是 多亚基复合物，由酶特异性多肽组成， 三种酶共有的亚基。人共同亚基（hRPB 5，hRPB 6， hRPB 8、hRPB 10-α和hRPB 10-β）是真正的通用转录 因为它们对所有人类基因的表达都是必不可少的。 三个HRP。因此，确定这些亚基的结构和功能 是阐明基本分子机制的重要一步， 控制着人类基因的转录。首席研究员已经 用核磁共振法测定了hRPB 6的溶液结构 (NMR)谱本提案侧重于以下方面的结构分析： 常见的人类亚基及其在分子生物学中的作用 人体RPII的结构和功能。具体目标和实验 本研究的方法是：1）进行高分辨率的 共同亚基hRPB 8，hRPB 10-α，hRPB 10-β， 使用X射线晶体学和核磁共振光谱学。2)确定 HRP共同亚基和一般亚基之间相互作用的结构基础 转录起始和延伸因子。为此，建议 进行与以下复合的hRPB 5亚基的X射线晶体学分析： 转录起始因子hTFIIB和hRPB 6亚基与 转录延伸因子hTFIIS。3)为了剖析 共同亚基hRPB 10 α、hRPB 10 β和hRPB 8在肿瘤功能中的作用 抑制蛋白 这些研究将提供高分辨率的三维结构， 人类共同亚基，并将阐明其在组装中的作用， 三个核hRPs的结构和功能。的原子坐标 这些共同的亚基，除了它们的科学价值，还可以用于 基于结构的化学品的合理设计，可以选择性地改变 hRPs的结构和功能，以及在分子生物学中的潜在应用 药

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanisms of the transcriptional regulation of gent expression at the molecular level. Transcription of all eukaryotic nuclear genes is performed by three distinct RNI polymerases (RPs) I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. RPs are multi-subunit complexes, consisting of enzyme-specific polypeptides and subunits common to all three enzymes. The human common subunits (hRPB5, hRPB6, hRPB8, hRPB10-alpha, and hRPB10-beta) are bona fide universal transcription factors because they are essential for the expression of all human genes by the three hRPs. Therefore, determining the structure and function of these subunits is an essential step towards elucidating the basic molecular mechanisms that control global human gene transcription. The principal investigator has already determined the solution structure o hRPB6 using Nuclear Magnetic Resonance (NMR) spectroscopy. The present proposal focuses on the structural analysis of the common human subunits and the elucidation of their roles in the molecular architecture and function of human RPII. The specific aims and experimental approaches of the proposed research are: 1) To perform a high-resolution structural analysis of the common subunits hRPB8, hRPB10-alpha, hRPB10-beta, using X-ray crystallography and NMR spectroscopy. 2) To determine the structural basis for the interactions between hRP common subunits and general transcription initiation and elongation factors. In this aim it is proposed to perform an X-ray crystallographic analysis of the hRPB5 subunit in complex with transcription initiation factor hTFIIB and of the hRPB6 subunit in complex with the transcription elongation factor hTFIIS. 3) To dissect the role of the common subunits hRPB10alpha, hRPB10beta, and hRPB8 in the function of tumor suppressor proteins. These studies will provide high-resolution three-dimensional structures of the human common subunits and will elucidate their roles in the assembly, structure, and function of the three nuclear hRPs. The atomic coordinates of the common subunits, besides their scientific value, could also be used for structure-based rational design of chemicals that could modify selectively the structure and function of the hRPs, with potential applications in molecular medicine.