Genetic studies on bacterial trans-translation

描述(申请人提供)：细菌拥有一套卓越的翻译质量控制系统。在被称为反式翻译的过程中，tmRNA进入停滞的核糖体，充当模板，编码一个短肽标签，标志着新生多肽的破坏。TmRNA直接从两个RNA模板合成单个多肽给我们对核糖体功能的理解提出了挑战。这项建议的目的是确定tmRNA、其蛋白质伙伴SmpB和核糖体清除停滞的mRNA、允许tmRNA进入并正确定位以恢复框架内翻译的机制。基因选择在相关的活体环境中快速分析大型突变体文库(数亿)的能力将被用于这三个组分的结构-功能研究，澄清它们之间的相互作用，以及识别负责翻译中未知活动的基因。该项目的具体目标是：1)使用一种新的基因选择，将细胞的寿命与tmRNA功能联系在一起，确定tmRNA中的三级结构和近端序列，以确保在tmRNA模板上的正确三联体处恢复翻译。2)将这种积极的选择应用于srnpB的研究，以确定负责欺骗解码机制允许tmRNA进入核糖体的序列和结构。这一活动所需的分子相互作用将通过恢复受伤的SmpB功能的进化遗传相互作用来确定。3)使用针对tmRNA功能的遗传选择(其中标签导致细胞死亡)，识别在停滞的核糖体中裂解mRNA的内切酶，为tmRNA进入扫清道路。我们的假设是，这种内切酶活性是核糖体本身的一种潜在功能。阐明翻译的机制将有助于深入了解核糖体在蛋白质合成中的重要功能。只在细菌中发现的转译机制也可能成为未来抗菌药物的靶标。

DESCRIPTION (provided by applicant): Bacteria possess a remarkable system for translational quality control. In a process known as trans-translation, tmRNA enters stalled ribosomes and acts as a template, encoding a short peptide tag that marks the nascent polypeptide for destruction. The tmRNA-directed synthesis of a single polypeptide from two RNA templates presents challenges to our understanding of ribosome function. The objective of this proposal is to determine the mechanism by which tmRNA, its protein partner SmpB, and the ribosome clear away the stalled mRNA, license tmRNA entry, and position it properly to resume translation in frame. The power of genetic selections to rapidly assay large libraries of mutants (hundreds of millions) in a relevant in vivo context will be brought to bear on structure-function studies of these three components, clarification of interactions between them, and the identification of genes responsible for uncharacterized activities in trans- translation. The specific aims of the project are: 1) Using a novel genetic selection that ties the life of the cell to tmRNA function, identify tertiary structures and proximal sequences in tmRNA that ensure translation resumes at the correct triplet on the tmRNA template. 2) Adapt this positive selection to the study of SrnpB to determine sequences and structures responsible for tricking the decoding machinery into allowing tmRNA into the ribosome. The molecular interactions required for this activity will be identified by evolving genetic interactions that restore wounded SmpB function. 3) Using a genetic selection against tmRNA function (in which tagging causes cell death), identify the endonuclease that cleaves mRNA inside stalled ribosomes, clearing the way for tmRNA entry. It is our hypothesis that this endonuclease activity is a latent function of the ribosome itself. Elucidation of the mechanism of trans-translation will yield insight into important aspects of ribosome function in protein synthesis. The trans-translation machinery, found only in bacteria, may also serve as a target for future antimicrobials.