Polysomes and Subunits: Structure-Function Relationship

描述（由申请人提供）：本补助金的长期目标 我们的建议是了解核糖体在原子水平上的功能， 蛋白质合成具体的目标包括开发一个遗传系统， 极端嗜热栖热菌Thermus thermophilus构建和分离突变体， rRNA和核糖体蛋白。形成交替的突变核糖体 翻译周期中的构象将被结晶化（在协作中 Jamie Cate和Venki Ramakrishnan博士）。第二个目标是调查 E.大肠杆菌核糖体，利用一系列的遗传和 我们实验室几年来开发的生化方法。主 一种方法涉及在质粒携带的rrn中构建rRNA突变， 操纵子各种菌株和载体甚至允许致命的突变， 与世隔绝30S和50S晶体研究提供的原子坐标 亚基将用于设计诱变策略以探测核糖体 在肽键形成、移位和 译码突变体形成稳定的功能性中间体将被表征 通过各种功能测定和cryoEM研究（与 Joachim Frank博士）。这些突变体中的一些将被构建并表达在 用于晶体研究的嗜热栖热菌。第三个目标涉及应用程序 这些相同的方法来探索的作用机制和阻力 影响蛋白质合成的抗生素。这些研究将提供 关于核糖体功能和构象变化的基本见解， rRNA和核糖体蛋白参与tRNA的选择、易位、肽 亚基内和亚基之间的键形成和信号传递。

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. Specific goals include developing a genetic system in the extreme thermophile Thermus thermophilus to construct and isolate mutants in rRNA and ribosomal proteins. Mutant ribosomes which form alternate conformations in the translation cycle will be crystalized (in collaboration with Drs. Jamie Cate and Venki Ramakrishnan). A second goal is to investigate dynamic aspects of E. coli ribosomes by utilizing a series of genetic and biochemical methods developed in our laboratory over several years. The primary approach involves the construction of rRNA mutations in a plasmid-borne rrn operon. A variety of strains and vectors permit even lethal mutations to be isolated. The atomic coordinates provided by crystal studies of the 30S and 50S subunits will be used in designing mutagenic strategies to probe ribosome structure and function during peptide bond formation, translocation and decoding. Mutants forming stable functional intermediates will be characterized by a variety of functional assays and studied by cryoEM (in collaboration with Dr. Joachim Frank). Some of these mutants will be constructed and expressed in Thermus thermophilus for crystal studies. A third goal involves the application of these same methods to probe the mechanism of action and resistance of antibiotics affecting protein synthesis. These studies should provide fundamental insights about ribosome function and conformational changes in the rRNA and ribosomal proteins involved in tRNA selection, translocation, peptide bond formation and signal transmission within and between the subunits.