Genetic studies on bacterial trans-translation

性状（由申请人提供）：细菌具有显著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模板合成单一多肽的tRNA指导提出了挑战，我们的核糖体功能的理解。本提案的目的是确定tmRNA、其蛋白伴侣SmpB和核糖体清除停滞的mRNA、许可tmRNA进入并将其适当定位以恢复框内翻译的机制。在相关的体内环境中快速测定大的突变体文库（数亿）的遗传选择的能力将对这三种组分的结构-功能研究、它们之间的相互作用的澄清以及负责反式翻译中的未表征活性的基因的鉴定产生影响。该项目的具体目标是：1）使用将细胞寿命与tmRNA功能联系起来的新型遗传选择，确定tmRNA中的三级结构和近端序列，以确保翻译在tmRNA模板上的正确三联体处恢复。2)将这种正选择应用于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。该活动所需的分子相互作用将通过进化恢复受损SmpB功能的遗传相互作用来确定。3)使用针对tmRNA功能的遗传选择（其中标记导致细胞死亡），鉴定在停滞的核糖体内切割mRNA的核酸内切酶，为tmRNA进入扫清道路。我们假设这种核酸内切酶活性是核糖体本身的潜在功能。反式翻译机制的阐明将有助于深入了解蛋白质合成中核糖体功能的重要方面。这种仅在细菌中发现的反式翻译机制也可能成为未来抗菌剂的靶点。

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.