Molecular Mechanism of Translation

描述(申请人提供)：蛋白质合成是所有生物的基本过程。在蛋白质合成的延伸阶段，核糖体准确地选择与mRNA密码子对应的氨基酰tRNA。这项建议的长期目标是研究核糖体选择tRNA的机制。结构研究表明，在tRNA选择过程中，核糖体识别密码子-反密码子双链的形状。然而，这些接触对tRNA选择和核糖体准确性的相对贡献尚不清楚。因此，这一建议的主要目标是确定核糖体和mRNA-tRNA复合体之间的特定接触在tRNA选择中的贡献。我们还将研究错误编码的抗生素如何影响tRNA选择过程。我们开发了一种新的、基于荧光的、稳定前的动力学分析方法，用于研究tRNA的选择。这一强大的方法将使我们能够确定核糖体中的哪些接触对tRNA选择最重要。此外，这项建议将使用猝灭流动方法、关键残基的定点突变和生化分析来研究核糖体选择tRNA的机制。这些实验将提供有关tRNA选择动力学的信息，这是X射线结晶学不容易获得的。 核糖体是几类抗生素灭活的靶标。抗生素如帕罗霉素、链霉素、四环素和新霉素会影响核糖体对tRNA的选择。链霉素用于治疗结核病，四环素用于治疗炭疽感染。耐抗生素的细菌菌株正在上升，在世界各地对这些感染的管理和治疗造成了危机。了解翻译的机制将为开发更有效的抗生素提供见解，这些抗生素针对这些耐药菌株的核糖体和传染性生物恐怖主义制剂。此外，这一提议将对RNA催化、生命起源、RNA结构和功能、真核蛋白质合成机制、癌症和神经科学等领域产生广泛影响。

DESCRIPTION (provided by applicant): Protein synthesis is a fundamental process in all living organisms. During the elongation phase of protein synthesis, the ribosome accurately selects the aminoacyl-tRNA corresponding to the mRNA codon. The long-term objective of this proposal is to study the mechanism of tRNA selection by the ribosome. Structural studies revealed that the ribosome recognizes the shape of the codon-anticodon duplex during tRNA selection. However, the relative contributions of these contacts toward tRNA selection and ribosomal accuracy are not known. Therefore, the major goal of this proposal is to determine the contribution of specific contacts between the ribosome and the mRNA-tRNA complex in tRNA selection. We will also study how miscoding antibiotics affect the process of tRNA selection. We have developed a new, fluorescence-based, pre-steady state kinetic assay for studying tRNA selection. This powerful method will permit us to determine which contacts within the ribosome are most important for tRNA selection. In addition, this proposal will use quench-flow methods, site-directed mutagenesis of critical residues, and biochemical assays to study the mechanism of tRNA selection by the ribosome. These experiments will provide information about the dynamics of tRNA selection that cannot be easily acquired by X-ray crystallography. Ribosomes are the target for inactivation by several classes of antibiotics. Antibiotics such as paromomycin, streptomycin, tetracycline, and neomycin affect tRNA selection by the ribosome. Streptomycin is used to treat tuberculosis and tetracycline is used to treat anthrax infections. Antibiotic-resistant strains of bacteria are on the rise, causing a crisis in the management and treatment of these infections throughout the world. Understanding the mechanism of translation will provide insights for developing more effective antibiotics that target the ribosome of these drug-resistant strains of bacteria and infectious bioterrorism agents. Furthermore, this proposal will have a broad impact on fields such as RNA catalysis, origin of life, RNA structure and function, mechanism of eukaryotic protein synthesis, cancer, and neuroscience.