Quality Control of Protein Translation

描述(申请人提供)：基因信息从DNA到RNA再到蛋白质的准确流动对所有活着的有机体都是必不可少的。一系列令人惊讶的质量保证机制已经发展起来，以确保在这一过程的每个阶段都保持高度保真。最吸引人的质量控制机制之一涉及tmRNA，也被称为SsrA或10Sa RNA。TmRNA是一种多功能的高度保守的细菌分子，具有tRNA和mRNA的结构和功能特性。我们以前的研究表明，所有已知的tmRNA活性都需要SmpB，SmpB是一种小蛋白，可以特异性地与tmRNA结合，并具有高亲和力，以促进其与停滞的核糖体的结合。SmpB-tmRNA系统协调三个关键的生物学功能：1)识别和挽救停滞在异常mRNAs上的核糖体，2)处理引起缺陷的mRNAs，以及3)在不完整的蛋白质片段上增加一个降解标签用于定向蛋白分解。虽然tmRNA活性在大肠杆菌中不是必需的，但对于细菌在不利条件下的生存和对某些甚至所有致病菌的毒力来说是必不可少的。我们实验室的最新证据表明，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只在原核生物中发现，涉及新的RNA和蛋白质因子，对于病原菌的生存是必不可少的，对这一系统的更深层次的机制了解可能会使设计高度特异的新抗菌剂成为可能。SmpB-tmRNA复合体的形成和随后对停滞的核糖体的识别的分子基础尚不清楚。本研究的目标是结合分子遗传学、蛋白质生物化学、生物信息学和结构学方法来阐明SmpB-tmRNA质量控制系统的作用机制。重点是SmpB-tmRNA复合体如何识别停滞的核糖体，并促进3‘-5’外切酶RNaseR对引起缺陷的mRNA的检测和选择性衰退的分子特征。具体地说，通过这些研究，我们希望了解SmpB和RNaseR与tmRNA和核糖体相互作用的生化和结构基础，即参与了哪些氨基酸残基，进行了哪些碱基特异性接触，哪些结构特征有助于形成与停滞的核糖体相关的SmpB和RNaseR复合体以及它们与停滞的核糖体的相互作用。与公共卫生相关：随着现有抗生素失效，采取新的应对措施的必要性变得更加迫切。这里概述的遗传、生化和结构研究提供了机会，以获得新的见解和更深入的机制了解一个独特的细菌监测系统由多功能的tmRNA和它的基本蛋白伙伴，SmpB。对这一特殊细菌系统的彻底了解，对于许多病原体的生存和毒力至关重要，应该为开发专门针对病原微生物的基于知识的新抗感染剂铺平道路。最终，这些洞察力将对更好地理解各种细胞过程产生影响，包括控制基因表达、蛋白质的合成和降解，以及有针对性的衰退缺陷mRNAs。

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.