Structure and Mechanism of Programmed Ribosomal Frameshifting in SARS coronavirus

说明(申请人提供)：冠状病毒(CoV)与严重疾病有关，2003年严重急性呼吸综合征(SARS)-CoV疫情证明了这一点。感染的关键步骤之一涉及病毒信使核糖核酸介导的基因表达的重新编码；在翻译过程中发生的A-1移码事件。正是这种优雅的机制使核糖体绕过了终止密码子，合成了病毒酶蛋白。此外，这种事件发生的频率对于有效的病毒感染性很重要， 并受翻译mRNA中的结构域调节(在SARS-CoV的情况下，该结构域是伪结)。虽然框架转移的各个方面已经得到了相当大的关注，但对影响效率的RNA结构和结构重排缺乏了解。我们的初步研究表明，逆转录病毒基因的表达受到活性、通读许可构象和非活性假结构象之间的质子驱动的动态平衡的调节。这一建议旨在通过结构研究与生化和活体实验相结合，对SARS-CoV的移码频率获得完整的结构和机制的了解。我们的目标将是：(#1)了解如何保持记录频率的基础，(#2)确定两种配置下的信使核糖核酸信号的结构：允许和不允许移帧，以及(#3)设计结构导向突变体来测试我们的平衡模型。

DESCRIPTION (provided by applicant): Coronaviruses (CoV) are associated with severe diseases as demonstrated by the 2003 severe acute respiratory syndrome (SARS)-CoV epidemic. One of the critical steps of infection involves viral mRNA mediated recoding of gene expression; a -1 frameshifting event that occurs during translation. It is this elegant mechanism that allows the ribosome to bypass a stop codon and synthesize viral enzymatic proteins. Furthermore, the frequency by which this event occurs is important for efficient viral infectivity, and is regulated by domains in the translating mRNA (in the case of the SARS-CoV, this domain is a pseudoknot). Although aspects of frameshifting have received considerable attention, an understanding of the RNA structures and structural rearrangements that influence the efficiency is lacking. Our preliminary studies indicate that retroviral gene expression is regulated by a dynamic, proton-driven equilibrium between an active, read-through permissive, and an inactive pseudoknot conformation. This proposal aims to gain a complete structural and mechanistic understanding of the frameshifting frequency in SARS-CoV by combining structural studies with biochemical and in vivo experiments. Our aims will be: (#1) to understand the basis for how the recoding frequency is maintained, (#2) to determine the structures of the mRNA signal in both configurations: permissive and non-permissive for frameshifting and (#3) to engineer structure-guided mutants to test our equilibrium model.