Genetic studies on bacterial trans-translation

细菌翻译的遗传学研究

• 批准号: 7409728
• 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/7409728
• 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; Peptide Elongation Factor Tu; 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; human EEF1A1 protein; 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）：细菌具有转化质量控制的显着系统。在称为跨翻译的过程中，TMRNA进入停滞的核糖体并充当模板，编码一个标记新生多肽的短肽标签，以破坏。来自两个RNA模板的单个多肽的TMRNA指导的合成对我们对核糖体功能的理解提出了挑战。该提案的目的是确定TMRNA，其蛋白质伴侣SMPB和核糖体清除失速mRNA，许可证TMRNA进入的机制，并将其适当地定位在框架中。遗传选择在相关的体内环境中快速测定突变体的大型文库（数亿）的力量将受到这三个组件的结构功能研究，澄清它们之间的相互作用以及对跨翻译中未表征活性的基因的识别。该项目的具体目的是：1）使用将细胞的寿命与TMRNA功能联系起来的新型遗传选择，识别TMRNA中的第三纪结构和近端序列，以确保在TMRNA模板上正确的Triplet恢复翻译。 2）将这种阳性选择适应SRNPB的研究，以确定负责欺骗解码机械使TMRNA进入核糖体的序列和结构。该活性所需的分子相互作用将通过恢复受伤的SMPB功能的遗传相互作用来识别。 3）使用针对TMRNA功能的遗传选择（其中标记会导致细胞死亡），确定核糖体内切割mRNA的内切酶，从而为TMRNA的进入扫清了道路。我们的假设是，这种核酸酶活性是核糖体本身的潜在功能。阐明跨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。仅在细菌中发现的跨翻译机制也可能是未来抗菌剂的靶标。