Polysomes & Subunits: Structure-Function Relationships

描述（由申请人提供）：本资助计划的长期目标是了解核糖体在蛋白质合成过程中如何在原子水平上发挥作用。我们的方法包括在极端嗜热栖热菌Thermus thermophilus中开发遗传和生化系统，以构建和分离rRNA，核糖体蛋白和翻译因子的突变体。我们的第一个目标是表征野生型T。通过结构探测在溶液中对嗜热核糖体进行修饰，并通过质谱法确定修饰的残基。这将作为随后分析rRNA和核糖体蛋白突变体的基线。将鉴定选定的突变体用于结晶尝试。第二个目的是研究核糖体和延伸因子EF-Tu和EF-G之间的相互作用的性质，在住宿和易位。将探测两个亚基中的选定功能位点。将评价易位过程中构象变化的模型以及放线菌素结核菌素抗生素抑制易位的机制。第三个目标是探索我们收集的T.嗜热菌的功能，以表征除了与该区域相关的肽键形成，包括易位，解码和肽释放。快速动力学实验将用于评估突变体和测试的适应模型，肽抗生素阴霉素将被检查其在终止中的作用。这些实验应该为核糖体结构与功能关系、翻译过程中核糖体的不同构象状态以及临床有用抗生素的作用模式和耐药性提供基本见解。

DESCRIPTION (provided by applicant): The long term objective of this grant proposal is to understand how the ribosome functions at the atomic level during protein synthesis. Our approach includes developing genetic and biochemical systems in the extreme thermophile Thermus thermophilus to construct and isolate mutants in rRNA, ribosomal proteins and translation factors. Our first aim is to characterize wild-type T. thermophilus ribosomes in solution by structure probing and determine modified residues by mass spectrometry. This will serve as the baseline for subsequent analysis of mutants in rRNA and ribosomal proteins. Selected mutants will be identified for crystallization attempts. A second aim is to investigate the nature of interactions between the ribosome and elongation factors EF-Tu and EF-G during accommodation and translocation. Selected functional sites in both subunits will be probed. A model for conformational change during translocation will be evaluated as will the mechanism of translocation inhibition by tuberactinomycin antibiotics. A third aim will probe our collection of mutants in the peptidyltransferase active site of 23S rRNA of T. thermophilus to characterize functions in addition to peptide bond formation associated with this region, including translocation, decoding and peptide release. Fast kinetic experiments will be used to evaluate the mutants and test a model of accommodation, and the peptide antibiotic negamycin will be examined for its role in termination. These experiments should provide fundamental insights into ribosome structure-function relationships, different conformational states of the ribosome during translation and the mode of action of and resistance to clinically useful antibiotics.