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全面了解生物作用和