STRUCTURE/FUNCTION STUDIES OF T7 RNA POLYMERASE

T7 RNA 聚合酶的结构/功能研究

• 批准号: 2900837
• 负责人: RUI J. SOUSA
• 金额: $ 21.08万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2000-12-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900837
• 关键词: DNA directed RNA polymerase; X ray crystallography; bacteriophage T7; chemical structure function; enzyme activity; enzyme structure; enzyme substrate; gene deletion mutation; genetic promoter element; genetic transcription; mutant; nuclear runoff assay; nucleotides; plasmids; protein purification; site directed mutagenesis; structural biology; virus protein

The proposed work is aimed at increasing our understanding of structure- function relationships in bacteriophage T7 RNA polymerase (RNAP). Our structural and structure-function studies of T7 RNAP have provided us with its 3-dimensional structure and have identified within this structure the template binding cleft, the active site, a region involved in binding the rNTP, and a domain which binds the nascent RNA. Comparison of the T7 RNAP structure to other polymerases and integration of published structure- function studies of polymerases into this comparison makes it possible for us to develop the concept of modularity in polymerase structure-function organization. We have identified subdomains or 'modules' in T7 RNAP which are not a part of the structurally conserved polymerase domain. The functions of these additional modules are poorly understood. We will mutagenize these modules in order to determine their functions and to test functional hypotheses which have emerged from modeling studies. Our structural and structure-function studies of the extended thumb subdomain of T7 RNAP have shown that it is a flexible structure and that it functions to hold the ternary complex together during transcription (probably by wrapping around bound template). These studies have identified a putative hinge or 'knuckle' within the thumb which may underlie its flexibility. We will mutagenize this 'knuckle' to test this functional hypothesis. We will characterize the properties of a mutation which causes the polymerase to indiscriminately utilize both rNTPs and dNTPs in order to learn more about the mechanisms of substrate discrimination and to characterize the properties of this potentially useful research reagent. Finally, we plan to mutagenize a region in T7 RNAP likely to be responsible for substrate discrimination with the aim of changing the substrate specificity of T7 RNAP so that it will selectively utilize dNTPs. This work has two goals: 1. identification of the substrate discrimination elements through modification of substrate specificity, and 2. the engineering of a polymerase which synthesizes DNA but has the unique properties of an RNA polymerase (promoter specificity, de novo initiation, template unwinding). Such a novel enzyme could find application in genetic research and gene therapy protocols. Generally, it should be recognized that polymerases are critical enzymes in many applications: in clinical virology they are therapeutic targets; they are research tools in molecular biology; and they synthesize the nucleic acids that are the targets/agents of gene therapy and transgenic research. The ability to engineer or target polymerases could result in development of new therapeutics and research tools. Most of these developments cannot be anticipated, though we frame one potential application. To be able to carry out such engineering/targeting we need a deeper understanding of polymerase structure-function relationships. Our work with T7 RNAP is aimed at contributing to such enhanced understanding.

The proposed work is aimed at increasing our understanding of structure- function relationships in bacteriophage T7 RNA polymerase (RNAP). Our structural and structure-function studies of T7 RNAP have provided us with its 3-dimensional structure and have identified within this structure the template binding cleft, the active site, a region involved in binding the rNTP, and a domain which binds the nascent RNA. Comparison of the T7 RNAP structure to other polymerases and integration of published structure- function studies of polymerases into this comparison makes it possible for us to develop the concept of modularity in polymerase structure-function organization. We have identified subdomains or 'modules' in T7 RNAP which are not a part of the structurally conserved polymerase domain. The functions of these additional modules are poorly understood. We will mutagenize these modules in order to determine their functions and to test functional hypotheses which have emerged from modeling studies. Our structural and structure-function studies of the extended thumb subdomain of T7 RNAP have shown that it is a flexible structure and that it functions to hold the ternary complex together during transcription (probably by wrapping around bound template). These studies have identified a putative hinge or 'knuckle' within the thumb which may underlie its flexibility. We will mutagenize this 'knuckle' to test this functional hypothesis. We will characterize the properties of a mutation which causes the polymerase to indiscriminately utilize both rNTPs and dNTPs in order to learn more about the mechanisms of substrate discrimination and to characterize the properties of this potentially useful research reagent. Finally, we plan to mutagenize a region in T7 RNAP likely to be responsible for substrate discrimination with the aim of changing the substrate specificity of T7 RNAP so that it will selectively utilize dNTPs. This work has two goals: 1. identification of the substrate discrimination elements through modification of substrate specificity, and 2. the engineering of a polymerase which synthesizes DNA but has the unique properties of an RNA polymerase (promoter specificity, de novo initiation, template unwinding). Such a novel enzyme could find application in genetic research and gene therapy protocols. Generally, it should be recognized that polymerases are critical enzymes in many applications: in clinical virology they are therapeutic targets; they are research tools in molecular biology; and they synthesize the nucleic acids that are the targets/agents of gene therapy and transgenic research. The ability to engineer or target polymerases could result in development of new therapeutics and research tools. Most of these developments cannot be anticipated, though we frame one potential application. To be able to carry out such engineering/targeting we need a deeper understanding of polymerase structure-function relationships. Our work with T7 RNAP is aimed at contributing to such enhanced understanding.