Molecular Mechanism and Biological Function of 3'-5' Polymerases

3-5聚合酶的分子机制和生物学功能

• 批准号: 9229039
• 负责人: Jane Elizabeth Jackman
• 金额: $ 34.34万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9229039
• 关键词: Affect; Antifungal Agents; Antiparasitic Agents; Archaea; Bacteria; Base Pairing; Biochemical; Biochemical Genetics; Biological; Biological Assay; Biological Process; Biology; Bypass; Catalysis; Cells; Chemicals; Coupled; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Defect; Development; Diabetic Nephropathy; Dictyostelium discoideum; Discrimination; Enzymes; Eukaryota; Exhibits; Family; Family member; Genetic; Goals; Growth; Health; Histidine-Specific tRNA; Human; Human Pathology; Investigation; Kinetics; Lead; Length; Life; Link; Metabolism; Microbe; Mitochondria; Molecular; Nature; Nucleic Acids; Nucleotides; Organism; Outcome; Pathway interactions; Phenotype; Physarum polycephalum; Plasmodium falciparum; Polymerase; Positioning Attribute; Process; Property; Protein Family; Proteins; RNA; RNA Editing; RNA Processing; RNA chemical synthesis; Reaction; Ribosomal RNA; Role; Saccharomyces cerevisiae; Specificity; Structure; Substrate Specificity; System; Techniques; Testing; Transfer RNA; Trichomonas vaginalis; Untranslated RNA; Variant; Yeasts; biological systems; genetic approach; guanylyltransferase; insight; knock-down; member; novel; overexpression; pathogen; preference; repaired; transcriptome sequencing; yeast genetics

The long term goals of this project are to develop a complete understanding of the biological roles and molecular mechanisms of the only known family of 3'-5' polymerases- enzymes that act in the opposite direction to all known DNA and RNA polymerases- in biology. The 3'-5' polymerase enzyme family contains tRNAHis guanylyltransferase (Thg1) proteins and Thg1-like proteins (TLPs). Thg1 proteins utilize the 3'-5' addition reaction to add a single required nucleotide to tRNAHis, which is an essential activity in many eukaryotes, including humans. On the other hand, although they share a related structure and basic catalytic mechanism, TLPs are biochemical and biologically distinct from Thg1, and the biological reactions that these enzymes catalyze are much less well-understood. At least one function of TLPs is to utilize Watson-Crick base pair dependent 3'-5' polymerase activity to add multiple nucleotides to repair the 5'-ends of tRNA in the mitochondria of many eukaryotic microbes. However, additional functions for these enzymes, including acting to repair or process other types of RNAs, are likely. Since RNA repair reactions are biologically important, and defects in these pathways can lead to negative effects on health, it is critical to fully understand the contributions of these unusual proteins to maintaining a healthy RNA pool. Interestingly, structures of several 3'-5' polymerases that are now available indicate that these enzymes share a distinct structural similarity and several aspects of their catalytic mechanism with canonical 5'-3' polymerases. Therefore, understanding the molecular basis for catalysis by 3'-5' polymerases is also important to understanding the distinctions between these two classes of nucleic acid synthesizing enzymes. The specific aims of this proposal are to determine biological roles of 3'-5' polymerases in the slime mold, Dictystelium discoideum, as well as in some Archaea and S. cerevisiae. The molecular basis for substrate recognition, which is a key biological property that distinguishes Thg1 and TLPs, will also be investigated. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5' addition reactions catalyzed by diverse 3'-5' polymerase family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5' nucleotide addition in biological systems.

该项目的长期目标是发展对生物学作用的全面理解， 唯一已知的3 '-5'聚合酶家族的分子机制-相反作用的酶 生物学中所有已知的DNA和RNA聚合酶的方向。3 '-5'聚合酶家族包含 tRNAHis鸟苷酰转移酶（Thg 1）蛋白和Thg 1样蛋白（TLPs）。Thg 1蛋白利用3 '-5'端的 在一些实施方案中，该方法包括将单个所需核苷酸添加到tRNAHis的加成反应，这在许多实施方案中是必需的活性。 真核生物，包括人类。另一方面，尽管它们具有相关的结构和基本的催化活性， 由于TLPs的作用机制，TLPs在生物化学和生物学上与Thg 1不同，这些TLPs的生物学反应与Thg 1的生物学反应不同。 催化酶的作用还不太清楚。TLP的至少一个功能是利用沃森-克里克碱 配对依赖性3 '-5'聚合酶活性，以添加多个核苷酸来修复细胞中tRNA的5 '末端。 许多真核微生物的线粒体。然而，这些酶的其他功能，包括作用于 来修复或加工其他类型的RNA。由于RNA修复反应在生物学上很重要， 这些途径中的缺陷可能导致对健康的负面影响，因此充分了解 这些不寻常的蛋白质对维持健康的RNA库的贡献。有趣的是，几种结构 现在可用的3 '-5'聚合酶表明这些酶具有明显的结构相似性， 它们与典型的5 ′-3 ′聚合酶的催化机制的几个方面。因此了解 3 '-5'聚合酶催化作用的分子基础对于理解 这两类核酸合成酶。本提案的具体目的是确定 3 '-5'聚合酶在黏菌、盘状网柄菌和一些放线菌中的生物学作用 和S.啤酒。底物识别的分子基础，这是一个关键的生物学特性， 区分Thg 1和TLPs，也将进行研究。本申请提出使用动力学的、遗传的、 生物化学和结构技术来研究两者的分子机制和生物学功能 由不同的3 '-5'聚合酶家族成员催化的非模板化和模板化3 '-5'加成反应。 这些结果将提供对一种新的、显然广泛存在的、但在很大程度上未被探索的， 反应，并将能够进一步研究3 '-5'核苷酸添加在生物学中的替代功能。 生物系统。