Molecular Mechanism of Translation

翻译分子机制

• 批准号: 7628610
• 负责人: SIMPSON JOSEPH
• 金额: $ 30.36万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7628610
• 关键词: Affect; Amino Acyl Transfer RNA; Anthrax disease; Antibiotic Resistance; Antibiotics; Anticodon; Bacteria; Biochemical; Biogenesis; Biological Assay; Bioterrorism; Catalysis; Codon Nucleotides; Complex; Drug resistance; Fluorescence; Goals; Infection; Kinetics; Life; Malignant Neoplasms; Messenger RNA; Methods; Molecular; Neomycin; Neurosciences; Organism; Paromomycin; Phase; Process; Protein Biosynthesis; RNA; Relative (related person); Ribosomes; Shapes; Site-Directed Mutagenesis; Streptomycin; Structure; Tetracyclines; Transfer RNA; Translations; Tuberculosis; X-Ray Crystallography; base; insight; research study; resistant strain

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.

描述（由申请人提供）：蛋白质合成是所有生物体的基本过程。 在蛋白质合成的延伸阶段，核糖体准确地选择与 mRNA 密码子相对应的氨酰基-tRNA。 该提案的长期目标是研究核糖体选择 tRNA 的机制。 结构研究表明，核糖体在 tRNA 选择过程中识别密码子-反密码子双链体的形状。 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，该提案的主要目标是确定核糖体和 mRNA-tRNA 复合物之间的特异性接触在 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前稳态动力学测定法，用于研究 tRNA 选择。 这种强大的方法将使我们能够确定核糖体内的哪些接触对于 tRNA 选择最重要。 此外，该提案将使用淬灭流方法、关键残基的定点诱变和生化测定来研究核糖体选择 tRNA 的机制。 这些实验将提供 X 射线晶体学无法轻易获得的 tRNA 选择动态信息。 核糖体是几类抗生素失活的目标。 巴龙霉素、链霉素、四环素和新霉素等抗生素会影响核糖体对 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。 抗生素耐药性细菌菌株正在增加，导致全世界这些感染的管理和治疗陷入危机。 了解翻译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细菌菌株和传染性生物恐怖剂的核糖体。 此外，该提案还将对RNA催化、生命起源、RNA结构与功能、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。