Polysomes & Subunits: Structure-Function Relationships

描述(由申请者提供)：这项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嗜热菌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通过结构探测来表征溶液中的野生型嗜热梭菌核糖体，并通过质谱分析来确定修饰残基。这将作为随后分析rRNA和核糖体蛋白突变的基线。选定的突变体将被确定用于结晶尝试。第二个目的是研究核糖体与伸长因子EF-TU和EF-G在调节和易位过程中相互作用的性质。将对两个亚基中选定的功能位点进行探测。我们将评估转位过程中构象变化的模型，以及结核放线菌素抑制转位的机制。第三个目标是探索我们收集的嗜热链霉菌23S rRNA肽基转移酶活性部位的突变株，以表征与该区域相关的肽键形成之外的功能，包括易位、解码和肽释放。快速动力学实验将被用来评估突变体和测试适应模型，多肽抗生素负霉素将被检测其在终止中的作用。这些实验应该提供对核糖体结构-功能关系、核糖体在翻译过程中的不同构象状态以及临床上使用的抗生素的作用模式和耐药性的基本见解。

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.