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Quality Control of Protein Translation

蛋白质翻译的质量控制

基本信息

批准号：
8305593
负责人：
A. WALI KARZAI
金额：
$ 30.47万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8305593
关键词：
Affinity Alanine-Specific tRNA Alanine-tRNA Ligase Amino Acids Anti-Bacterial Agents Anti-Infective Agents Antibiotics Anticodon Binding Biochemical Bioinformatics Biological Process C-terminal Cell physiology Charge Codon Nucleotides Complex DNA Data Detection Development Distal EEF1A1 gene Effectiveness Elements Ensure Escherichia coli Event Exoribonucleases Family Gene Expression Genetic Goals Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Health Hydrolysis Kinetics Laboratories Life Light Link Measures Mediating Messenger RNA Molecular Molecular Genetics Organism Pathway interactions Peptides Phosphodiesterase I Physiological Play Positioning Attribute Process Prokaryotic Cells Property Protein Binding Protein Biochemistry Protein Fragment Proteins Proteolysis Quality Control RNA RNA-Protein Interaction Reading Frames Relative (related person)Research Ribosomes Role Site SmpB protein Staging Structure Surface System Tail Transcript Transfer RNA Translation Process Translations Variant Virulence base design fascinate insight knowledge base mRNA Decay mRNA Stability member microorganism novel pathogenic bacteria programs protein degradation quality assurance ribonuclease R stem tmRNA

项目摘要

DESCRIPTION (provided by applicant): The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and an mRNA. Our previous studies have shown that all known activities of tmRNA require SmpB, a small protein that binds tmRNA specifically and with high affinity to promote its association with stalled ribosomes. The SmpB-tmRNA system orchestrates three key biological functions: 1) recognition and rescue of ribosomes stalled on aberrant mRNAs, 2) disposal of the causative defective mRNAs, and 3) addition of a degradation tag to the incomplete protein fragments for directed proteolysis. Although not essential in E. coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence from our laboratory suggests that in addition to its quality control function the tmRNA system might also play a key regulatory role in certain physiological pathways. Moreover, because the SmpB and tmRNA are found only in prokaryotes, involves novel RNA and protein factors, and is essential for the survival of pathogenic bacteria, a deeper mechanistic understanding of this system might allow the design of highly specific new anti-bacterial agents. The molecular basis for the formation of the SmpB-tmRNA complex and the subsequent recognition of stalled ribosomes are not well understood. The objective of this research program is to use a combination of molecular genetics, protein biochemistry, bioinformatics, and structural approaches to elucidate the mechanism of action of the SmpB-tmRNA quality control system. The emphasis is on the molecular characterization of how SmpB-tmRNA complex recognizes stalled ribosomes and promotes the detection and selective decay of the causative defective mRNA by the 3'-5' exonuclease RNase R. Specifically, through these studies we wish to understand the biochemical and structural basis for the interactions of SmpB and RNase R with tmRNA and the ribosome; i.e. what amino acid residues are involved, what base specific contacts are made, what structural features contribute to the formation of the tmRNA-associated SmpB and RNase R complexes and their interaction with stalled ribosome. PUBLIC HEALTH RELEVANCE: As currently available antibiotics lose their effectiveness the need for new counter measure becomes ever more urgent. The genetic, biochemical, and structural studies outlined here offer the opportunity to gain novel insights into and a deeper mechanistic understanding of a unique bacterial surveillance system mediated by the versatile tmRNA and its essential protein partner, SmpB. A thorough understanding of this extraordinary bacterial system, essential for survival and virulence of many pathogenic bacteria, should pave the way for development of knowledge-based new anti-infective agents that exclusively target pathogenic microorganisms. Ultimately, these insights will have implications for a better understanding of a variety of cellular processes, including control of gene expression, synthesis and degradation of proteins, and the targeted decay defective mRNAs.

描述（由申请人提供）：遗传信息从DNA到RNA再到蛋白质的准确流动对所有生物体都是必不可少的。一系列惊人的质量保证机制已经形成，以确保在这一过程的每个阶段都保持高度的保真度。最令人着迷的质量控制机制之一涉及tmRNA，也称为SsrA或10Sa RNA。tmRNA是一种多功能且高度保守的细菌分子，具有tRNA和mRNA的结构和功能特性。我们之前的研究表明，所有已知的tmRNA的活性都需要SmpB， SmpB是一种小蛋白，它以高亲和力特异性结合tmRNA，促进其与停滞核糖体的结合。SmpB-tmRNA系统协调了三个关键的生物学功能：1)识别和拯救在异常mrna上停滞的核糖体，2)处理致病缺陷mrna，以及3)在不完整的蛋白质片段上添加降解标签以进行定向蛋白质水解。虽然在大肠杆菌中不是必需的，但tmRNA的活性对于细菌在不利条件下的生存和某些甚至所有致病菌的毒力都是必不可少的。我们实验室最近的证据表明，除了其质量控制功能外，tmRNA系统还可能在某些生理途径中发挥关键的调节作用。此外，由于SmpB和tmRNA仅存在于原核生物中，涉及新的RNA和蛋白质因子，并且对致病菌的生存至关重要，因此对该系统的更深入的机制理解可能允许设计高度特异性的新型抗菌药物。SmpB-tmRNA复合物形成的分子基础以及随后对停滞核糖体的识别尚不清楚。本研究计划的目的是结合分子遗传学，蛋白质生物化学，生物信息学和结构方法来阐明SmpB-tmRNA质量控制系统的作用机制。重点是SmpB-tmRNA复合物如何识别停止的核糖体，并促进3‘-5’外切酶RNase R对致病缺陷mRNA的检测和选择性衰变的分子表征。具体而言，通过这些研究，我们希望了解SmpB和RNase R与tmRNA和核糖体相互作用的生化和结构基础；例如，哪些氨基酸残基参与其中，哪些碱基特异性接触产生，哪些结构特征有助于形成与tmrna相关的SmpB和RNase R复合物以及它们与停滞核糖体的相互作用。公共卫生相关性：由于目前可用的抗生素失去其有效性，对新的应对措施的需求变得更加迫切。本文概述的遗传、生化和结构研究为获得对由多功能tmRNA及其必需蛋白伙伴SmpB介导的独特细菌监视系统的新见解和更深入的机制理解提供了机会。对这种对许多致病菌的生存和毒力至关重要的特殊细菌系统的彻底了解，应该为开发专门针对致病菌的基于知识的新型抗感染药物铺平道路。最终，这些见解将对更好地理解各种细胞过程产生影响，包括基因表达的控制，蛋白质的合成和降解，以及靶向衰变缺陷mrna。