Genetic studies on bacterial trans-translation

描述（申请人提供）：细菌具有卓越的翻译质量控制系统。在一个被称为反式翻译的过程中，tmRNA进入停滞的核糖体并作为模板，编码一个短肽标签，标记新生多肽的破坏。从两个RNA模板中合成单个多肽的tmrna对我们对核糖体功能的理解提出了挑战。本研究的目的是确定tmRNA及其蛋白伙伴SmpB和核糖体清除停滞mRNA的机制，允许tmRNA进入，并将其正确定位以恢复在框架中的翻译。在相关的体内环境中，基因选择能够快速测定大量突变体文库（数以亿计），这将对这三种成分的结构-功能研究产生影响，澄清它们之间的相互作用，并确定负责反翻译中未表征活动的基因。该项目的具体目标是：1)使用一种新的遗传选择，将细胞的生命与tmRNA的功能联系起来，确定tmRNA的三级结构和近端序列，确保翻译在tmRNA模板上的正确三联体恢复。2)将这种阳性选择应用于SrnpB的研究，以确定负责欺骗解码机制使tmRNA进入核糖体的序列和结构。这种活性所需的分子相互作用将通过进化的基因相互作用来确定，从而恢复受损的SmpB功能。3)利用针对tmRNA功能的遗传选择（其中标记导致细胞死亡），鉴定在停滞核糖体内切割mRNA的内切酶，为tmRNA的进入扫清道路。我们的假设是，这种内切酶活性是核糖体本身的潜在功能。反翻译机制的阐明将有助于深入了解核糖体在蛋白质合成中的重要功能。仅在细菌中发现的反翻译机制也可能作为未来抗菌剂的靶标。

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.