CRYSTAL STRUCTURE DETERMINATION OF QUEOSINE-SYNTHESIS AND OTHER TRNA MODIFICATIO

奎辛合成和其他 TRNA 修饰的晶体结构测定

• 批准号: 7954428
• 负责人: MANAL A SWAIRJO
• 金额: $ 0.21万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954428
• 关键词: Anabolism; Antibiotics; Anticodon; Automation; Biochemical; Cells; Codon Nucleotides; Complex; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Data; Development; Drug Delivery Systems; Enzymes; Funding; Genomics; Grant; HIV; Institution; Ligands; Methods; Modification; Pathway interactions; Pharmacologic Substance; Prokaryotic Cells; Research; Research Personnel; Resources; Ribosomes; Shigella Infections; Source; Structure; Technology; Time; Transfer RNA; Translations; United States National Institutes of Health; analog; beamline; comparative; structural biology; synchrotron radiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have used SSRL beam time on this proposal to conduct crystallographic studies on enzymes responsible for the biosynthesis of two tRNA modifications: queosine (Q) and threonyl carbamoyladenosine (t6A). These are two modifications of the tRNA anticodon loop that alter codon definitions and fine tune ribosome function during translation. Q biosynthesis is an established drug target for anti-shigellosis antibiotics. Similarly, due to its requirement for HIV replication, t6A modification of host-cell tRNA has been proposed as a potential target for the development of anti-HIV pharmaceuticals. Using a combination of comparative genomic, biochemical, and structural methods, we identified enzymes involved in the biosynthesis of Q and t6A. Using SSRL x-rays, we determined the structure of GCYH-IB, our newly discovered and first enzyme in the Q biosynthesis pathway in prokaryotes, in complex with a substrate analog and we collected native and heavy-atom derivative data for YkvM, the enzyme that catalyzes the third step in Q biosynthesis. We also determined the crystal structure of YrdC, a universally conserved and essential enzyme involved in t6A biosynthesis, in complex with its ATP substrate. In this renewal, we plan to start crystallographic studies of GCYH-IBinhibitor complexes and of QueA, which catalyzes a late step in Q biosynthesis, in complex with its tRNA substrate. We currently have crystals of GCYH-IB-ligand complexes and crystallization of the latter complex is underway. We plan to continue to use the SSRL beamline automation technology and remote access for these studies.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们在此提案中使用了 SSRL 光束时间，对负责两种 tRNA 修饰生物合成的酶进行晶体学研究：奎奥辛 (Q) 和苏氨酰氨基甲酰基腺苷 (t6A)。这是 tRNA 反密码子环的两种修饰，可改变密码子定义并在翻译过程中微调核糖体功能。 Q生物合成是抗志贺菌病抗生素的既定药物靶点。同样，由于 HIV 复制的需要，宿主细胞 tRNA 的 t6A 修饰已被提议作为开发抗 HIV 药物的潜在靶标。通过结合比较基因组、生物化学和结构方法，我们鉴定了参与 Q 和 t6A 生物合成的酶。使用 SSRL X 射线，我们确定了 GCYH-IB 的结构，GCYH-IB 是我们新发现的原核生物 Q 生物合成途径中的第一种酶，与底物类似物复合，并且我们收集了 YkvM（该酶）的天然和重原子衍生物数据催化 Q 生物合成的第三步。我们还确定了 YrdC 的晶体结构，YrdC 是一种参与 t6A 生物合成的普遍保守且必需的酶，与其 ATP 底物复合。在这次更新中，我们计划开始 GCYH-IBinhibitor 复合物和 QueA 的晶体学研究，QueA 与其 tRNA 底物复合，催化 Q 生物合成的后期步骤。我们目前拥有 GCYH-IB-配体复合物的晶体，并且后者复合物的结晶正在进行中。我们计划继续使用 SSRL 光束线自动化技术和远程访问来进行这些研究。