Genetic studies on bacterial trans-translation

细菌翻译的遗传学研究

• 批准号: 7616746
• 负责人: Allen Rowdon Buskirk
• 金额: $ 21.85万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616746
• 关键词: Address; Alanine; Amino Acids; Amino Acyl Transfer RNA; Bacteria; Binding; Biological Assay; C-terminal; Cell Death; Cells; Cleaved cell; Codon Nucleotides; EEF1A1 gene; Elements; Ensure; Eubacterium; Functional RNA; Future; Genes; Genetic; Genome; Goals; In Vitro; Left; Libraries; Licensing; Life; Link; Messenger RNA; Methodology; Molecular Target; Mutagenesis; Peptides; Positioning Attribute; Process; Protein Biosynthesis; Proteins; Quality Control; RNA; RNA, Ribosomal, 16S; Recycling; Research Personnel; Ribosomal RNA; Ribosomes; Role; Site; SmpB protein; Structure; System; Tail; Translation Process; Translations; Triplet Multiple Birth; Ursidae Family; antimicrobial; endonuclease; genetic selection; in vivo; insight; mutant; novel; polypeptide; programs; tmRNA; tool

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.

描述（由申请人提供）：细菌拥有卓越的翻译质量控制系统。在一个称为反式翻译的过程中，tmRNA 进入停滞的核糖体并充当模板，编码一个短肽标签，标记新生多肽以进行破坏。从两个 RNA 模板合成单个多肽的 tmRNA 指导对我们理解核糖体功能提出了挑战。该提案的目的是确定 tmRNA、其蛋白质伴侣 SmpB 和核糖体清除停滞的 mRNA、允许 tmRNA 进入并将其正确定位以恢复框架内翻译的机制。遗传选择在相关体内环境中快速分析大型突变体文库（数亿个）的能力将应用于这三个成分的结构功能研究，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) 使用一种新颖的遗传选择，将细胞的生命与 tmRNA 功能联系起来，识别 tmRNA 中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处恢复。 2) 将这种正向选择应用于 SrnpB 的研究，以确定负责欺骗解码机制以允许 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恢复受损 SmpB 功能的进化遗传相互作用来确定。 3) 利用针对 tmRNA 功能（其中标记导致细胞死亡）的遗传选择，识别在停滞核糖体内切割 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。阐明反式翻译机制将深入了解蛋白质合成中核糖体功能的重要方面。仅在细菌中发现的转译机制也可能作为未来抗菌药物的目标。