RNA Modification Enzymes: Methyltransferase/psi-synthase

RNA 修饰酶：甲基转移酶/psi-合酶

• 批准号: 7220658
• 负责人: JANET S. FINER-MOORE
• 金额: $ 27.01万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7220658
• 关键词: 5 fluorouridine; Alkylation; Amino Acids; Anti-HIV Agents; Bacillus (bacterium); Biochemical; Catalysis; Cells; Chemicals; Cocrystallography; Code; Complex; Crystallization; Enzymes; Escherichia coli; Evaluation; Exhibits; Family; Genes; Goals; HIV; Human; Iron; Kinetics; Length; Life; Measurement; Methyltransferase; Modification; Mutagenesis; Mutate; Mutation; Nucleotides; Oligonucleotides; Orthologous Gene; Pathway interactions; Physiological; Positioning Attribute; Proteins; Pseudouridine; Pyrimidine; Pyrimidines; RNA; RNA Folding; RNA-Protein Interaction; Relative (related person); Ribonucleotides; Ribosomal RNA; Ribosomes; Roentgen Rays; Role; S-Adenosylmethionine; Site; Small RNA; Specificity; Staging; Structure; Sulfur; Thermotoga maritima; Transfer RNA; Uracil; X-Ray Crystallography; base; cofactor; design; drug development; drug discovery; enzyme mechanism; enzyme structure; improved; knockout gene; member; mutant; p65; research study; success

DESCRIPTION (provided by applicant): Through all kingdoms of life RNAs are modified as, or after, they are synthesized. The types and sites of modification are often conserved, implying conservation of function. Approximately 0.8% of the coding capacity of the E. coli cell is dedicated to encoding enzymes that modify RNA. Many of these modifications are located at key functional regions of the ribosome or other RNAs. The enzymes that carry out the modifications exhibit unique or limited multisite specificity. The aim of this proposal is to determine the basis for selectivity and catalysis in three families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases), 5-methyl cystosine (mSC) MTases, and pseudouridine synthases (Psi synthases). These families evoke some of the most common conserved modifications of the more than 80 different types seen in RNA. A common feature of the chemical mechanisms of these families is Michael addition of an amino acid nucleophile to C-6 of the target pyrimidine to activate C-5 for alkylation. Each modification is evoked at a particular stage in RNA folding suggesting that the determinants of selectivity often involve unique three-dimensional folded structures. We seek to determine X-ray crystal structures of several members of each family to elucidate the basis for three-dimensional selectivity in enzyme-RNA targeting. The strategy is to express each enzyme from E. coli and orthologs from two other species, B. subtilis and T. maritima, to provide many candidates for crystallization trials. A minimal, often 17-50 base RNA substrate is sought. A synthetic minimal substrate, containing 5-fluorofoyrimidine at the target site, when reacted with the protein yields a covalent protein-substrate complex, providing an effective means to crystallize enzyme-RNA complexes. The structures of these are compared to structures of enzymes alone or with cofactors. The structures are the basis for the design of mutations in the enzyme or the substrate, and for computational evaluations. Biochemical and structural analysis of the mutant enzymes or substrates further elucidate contributors to the high selectivity and enzyme mechanism. An unrelated RNA MTase, human m1A58 tRNA MTase, will also be subject to structural determination to facilitate structure-based drug discovery. This is a required enzyme in HIV replication, and as such it may represent a new target for anti-HIV drugs.

描述（由申请人提供）：通过生命的所有王国，RNA被修改为或之后，它们被合成。修饰的类型和位点通常是保守的，这意味着功能保存。大肠杆菌细胞的编码能力的大约0.8％用于编码修饰RNA的酶。这些修饰中的许多修饰位于核糖体或其他RNA的关键功能区域。进行修饰的酶具有独特或有限的多站点特异性。该提案的目的是确定三个修饰酶的RNA家族的选择性和催化的基础：5-甲基尿嘧啶（M5U）甲基转移酶（MTases），5-甲基半胱氨酸（MSC）MTase MTase MTase和pseudouridine合酶（PSI合酶）。这些家庭唤起了RNA中看到的80多种不同类型的最常见的保守修改。这些家族化学机制的一个共同特征是迈克尔在靶嘧啶的C-6中添加了氨基酸亲核试剂，以激活C-5进行烷基化。在RNA折叠的特定阶段唤起了每种修饰，这表明选择性的决定因素通常涉及独特的三维折叠结构。我们试图确定每个家族几个成员的X射线晶体结构，以阐明酶-RNA靶向三维选择性的基础。该策略是表达来自大肠杆菌和直系同源物的每种酶。寻找最小的，通常是17-50个碱基RNA底物。当与蛋白质反应时，含有5-氟酰亚胺的合成最小底物可产生共价蛋白质底物复合物，从而提供有效的手段来结晶酶-RNA复合物。将它们的结构与单独的酶或辅因子的结构进行比较。结构是设计酶或底物中突变的基础，以及计算评估。突变酶或底物的生化和结构分析进一步阐明了高选择性和酶机制的贡献者。 无关的RNA MTase人M1A58 tRNA MTase也将受到结构确定，以促进基于结构的药物发现。这是HIV复制中所需的酶，因此可能代表了抗HIV药物的新靶标。