Polysomes and Subunits: Structure-Function Relationship

描述(由申请人提供)：本基金的长期目标 建议是了解核糖体如何在原子水平上发挥作用 蛋白质合成。具体目标包括开发一种基因系统 极端嗜热嗜热菌构建和分离突变株的研究 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(在合作中 杰米·凯特博士和文基·拉马克里希南博士)。第二个目标是调查 利用一系列遗传和分子生物学技术研究大肠杆菌核糖体的动态变化 生化方法是我们实验室几年来发展起来的。初级阶段 该方法涉及构建质粒携带的rrn中的rrna突变。 歌剧。各种菌株和载体甚至允许致命的突变 与世隔绝。30年代和50年代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 多肽键形成、转移和释放过程中的结构和功能 解码。形成稳定的功能中间体的突变体将被表征 通过各种功能分析并通过CryoEM进行研究(与 约阿希姆·弗兰克博士)。其中一些突变体将被构建并在 用于晶体研究的嗜热嗜热菌。第三个目标涉及应用程序 这些相同的方法来探索作用和抵抗的机制 影响蛋白质合成的抗生素。这些研究应该提供 对核糖体功能和构象变化的基本见解 参与tRNA选择、转位、多肽的rRNA和核糖体蛋白 亚基内部和亚基之间的键形成和信号传输。

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.