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）：氨基酸-TRNA合成酶通过氨基酰基化反应建立遗传代码，这些反应将特定氨基酸与携带三重态二尖序序列的trnas联系起来。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开发特异性的最古老的蛋白质之一。伴半胱氨酸-TRNA合成酶（CYSRS）的特异性是非凡的 - 即使是用丝氨酸的羟基半胱氨酸的硫醇的简单替代，或者用丝氨酸的羟基替代，或者在tRNA Cys中的单个替代可能会导致一百万倍或更多倍的活性降低。尽管最近的研究为CYSR的底物特异性提供了重大见解，但这不足以理解更广泛的进化术语的特异性。例如，最近的研究已经确定了一个嵌入的CYSR，该CYSR嵌入了ProlyltRNA合成酶（PROR）的序列框架中，该酶具有激活脯氨酸和半胱氨酸的能力，并催化了用脯氨酸和半胱氨酸催化tRNA的氨基酰基化。双重特异性PROR挑战了一种合成酶对一种氨基酸的看法，并提出了许多有关合成酶特异性的基本问题。此外，通过间接读数结构特征的识别是针对细菌（但未）Cysrs的结构特征的。这样的间接读数可以对我们对特异性的理解产生重大影响，远远超出了对tRNA-合成酶相互作用中直接接触的分析而获得的。细菌和桉树识别之间的差异也为发展氨基酰化的物种特异性抑制剂提供了基础。此外，大肠杆菌CYSR的新兴晶体结构现在提供了一个新的机会，可以解决该酶如何识别半胱氨酸的杰出问题，并在没有编辑机制的情况下对紧密相似的丝氨酸进行区分。这项研究是及时的，并有望对靶向氨基酸代谢疾病的分子医学进行新的见解。提出了三个具体目的：（1）通过嗜卤代寄生的卤代卤代的双重特异性来研究对trnacys的识别，（2），研究tRNA结构性基础的间接读数的热力学和结构贡献，以研究其在氨基化和（3）氨基化的能力基础上（​​3），并（3）研究了氨基col的能力。丝氨酸的半胱氨酸。这些研究应为负责遗传密码准确翻译的分子相互作用提供新的启示。