Functional Analysis of an RNA Structural Motif

RNA 结构基序的功能分析

• 批准号: 6782677
• 负责人: Ya-Ming Hou
• 金额: $ 29.73万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6782677
• 关键词: Escherichia coli; Halobacteriaceae; SDS polyacrylamide gel electrophoresis; X ray crystallography; acylation; aminoacid tRNA ligase; aminoacyl tRNA; bacterial RNA; bacterial proteins; biochemical evolution; cysteine; enzyme activity; enzyme mechanism; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure function; site directed mutagenesis; structural biology; sugar phosphates; transfer RNA; uracil nucleotides

DESCRIPTION (provided by applicant): Aminoacy-tRNA synthetases establish the genetic code through aminoacylation reactions that link specific amino acids to tRNAs that bear triplet anticodon sequences. The universal distribution of these enzymes across the phylogenetic tree suggests that they are among the oldest proteins to have developed specificity towards amino acids and tRNAs. The specificity of cysteinyl-tRNA synthetase (CysRS) is extraordinary- even the simple replacement of the thiol of the substrate cysteine with the hydroxyl of serine, or a single substitution in tRNA cys can cause reduction in activity of a million-fold or more. Although recent studies have provided major insights into the substrate specificity of CysRS, this is not sufficient to understand specificity in broader evolutionary terms. For example, recent studies have identified a CysRS embedded within the sequence framework of a prolyltRNA synthetase (ProRS) that has the ability to activate both proline and cysteine and catalyze aminoacylation of tRNA with proline and with cysteine. The dual-specificity ProRS has challenged the view of one synthetase for one amino acid and raised many fundamental questions about synthetase specificity. In addition, recognition of tRNAcys through indirect readout of structural features has been established for bacterial, but not eucaryotic, CysRS. Such an indirect readout can have major impact on our understanding of specificity well beyond that obtained from analysis of direct contacts in tRNA-synthetase interactions. The difference between the bacterial and eucaryotic recognition also provides the basis for developing species-specific inhibitors of aminoacylation. Further, emerging crystal structures of E. coli CysRS have now offered a novel opportunity to address the outstanding question of how this enzyme recognizes cysteine and discriminates against the closely similar serine without an editing mechanism. This investigation is timely and promises new insight into molecular medicine that targets disorders in amino acid metabolism. Three specific aims are proposed: (1) to study recognition of tRNAcys by the dual-specificity ProRS of the halophilic archaeon Halobacteriurn halobium, (2) to study the thermodynamic and structural contribution of indirect readout of tRNA structural motifs in aminoacylation, and (3) to study the molecular basis of the exquisite specificity of E. coli CysRS for its ability to distinguish cysteine from serine. These studies shall shed new light on the molecular interactions responsible for the accurate translation of the genetic code.

描述（由申请人提供）：氨酰-tRNA合成酶通过氨酰化反应建立遗传密码，该反应将特定氨基酸与带有三联体反密码子序列的tRNA连接起来。这些酶在系统发育树上的普遍分布表明它们是对氨基酸和 tRNA 产生特异性的最古老的蛋白质之一。半胱氨酰-tRNA 合成酶 (CysRS) 的特异性非常出色，即使简单地用丝氨酸的羟基替换底物半胱氨酸的硫醇，或者 tRNA cys 中的单个取代，也会导致活性降低一百万倍或更多。尽管最近的研究对 CysRS 的底物特异性提供了重要见解，但这不足以理解更广泛的进化术语中的特异性。例如，最近的研究发现嵌入脯氨酰RNA合成酶（ProRS）序列框架内的CysRS能够激活脯氨酸和半胱氨酸并催化tRNA与脯氨酸和半胱氨酸的氨酰化。双特异性 ProRS 挑战了一种合成酶对应一种氨基酸的观点，并提出了许多关于合成酶特异性的基本问题。此外，通过间接读取结构特征来识别 tRNAcys 的方法已针对细菌（而非真核生物）的 CysRS 建立。这种间接读数对我们对特异性的理解产生的重大影响远远超出了通过分析 tRNA-合成酶相互作用中的直接接触所获得的影响。细菌和真核识别之间的差异也为开发物种特异性的氨酰化抑制剂提供了基础。此外，大肠杆菌 CysRS 的新兴晶体结构现在提供了一个新的机会来解决这种酶如何识别半胱氨酸并在没有编辑机制的情况下区分非常相似的丝氨酸的突出问题。这项研究非常及时，为针对氨基酸代谢紊乱的分子医学带来了新的见解。提出了三个具体目标：（1）研究嗜盐古菌 Halobacteriurn halobium 的双特异性 ProRS 对 tRNAcys 的识别，（2）研究氨酰化中 tRNA 结构基序间接读出的热力学和结构贡献，以及（3） ）研究大肠杆菌 CysRS 区分半胱氨酸和丝氨酸的能力的精细特异性的分子基础。这些研究将为遗传密码准确翻译的分子相互作用提供新的线索。