Manipulation of cellular translation machinery by viral RNAs

描述(申请人提供)：翻译起始是真核生物基因调控的主要目标。内部核糖体进入位点(IRES)是一种RNA序列，它驱动非规范的翻译启动机制，绕过了信使RNA(MRNA)上5‘端的需要。IRES是许多重要病毒(如丙型肝炎、甲型肝炎、脊髓灰质炎病毒、HIV-1)感染周期的核心角色，是基因表达的重要调节因子。许多IRESS拥有与翻译机器交互和操纵的更高级别的结构，但我们对它们如何工作的理解是初级的。因此，我们对使用IRESS的病毒的了解仍然不完整，我们利用IRESS作为药物靶点的能力有限。此外，由于已知一些IRESS直接与核糖体相互作用以改变其构象并操纵其功能，IRESS为了解核糖体如何工作、结构RNA如何与核糖体结合以及核糖体功能如何改变提供了一个窗口。Dicistroviridae的基因间(IGR)IRESS为研究这些现象提供了一个强大的模型系统。我们建议建立在我们对Dicistroviridae的基因间(IGR)IRESS的长期研究的基础上，该研究驱动了核糖体招募和激活的高度简化的模式。在过去的几年里，我们的发现导致了IGR IRES功能的基于结构的详细模型；我们现在准备测试该模型，并前所未有地详细解释IRES的功能。我们建议这样做有三个具体目标。首先，我们将使用单分子FRET(SmFRET)来观察IRES RNAs、tRNAs和核糖体组件在引发复合体中的运动。通过观察结构IRES如何驱动核糖体上的特定运动，并将这些运动与规范伸长的运动进行比较，我们将在尚未达到的水平上获得洞察力。其次，我们将探索IRES RNA结构本身的构象变化，将这些动态变化与启动复合体中的全球变化联系起来，并在这样做的过程中，将静态图像转变为动态路径。第三，我们将通过X射线结晶学来解决IRES-核糖体复合体的结构，这应该会揭示迄今为止未曾见过的密切的核糖体-IRES相互作用，这些相互作用是构象变化的基础。这三个独立但互补的目标有望产生一幅连贯、高分辨率和动态的机制图，展示IRES RNA如何操纵基本的生物机器，从而为普遍的生物过程提供广泛的见解。

DESCRIPTION (provided by applicant): Translation initiation is a major target of gene regulation in eukaryotes. Internal ribosome entry sites (IRES) are RNA sequences that drive a non-canonical mechanism of translation initiation, bypassing the need for a 5'cap on the messenger RNA (mRNA). IRESs are central players in the infection cycles of many important viruses (e.g. hepatitis C, hepatitis A, poliovirus, HIV-1) and are significant regulators of gene expression. Many IRESs possess higher-order structures that interact with and manipulate the translation machinery, but our understanding of how they work is rudimentary. Thus, our knowledge of the viruses that employ IRESs remains incomplete, and our ability to exploit IRESs as drug targets is limited. Furthermore, because some IRESs are known to interact directly with the ribosome to alter its conformation and manipulate its function, IRESs offer a window into how ribosomes work, how structured RNAs can bind to ribosomes, and how ribosome function can be altered. The intergenic (IGR) IRESs of the Dicistroviridae provide a powerful model system to explore these phenomena. We propose to build upon our long-standing studies on the intergenic (IGR) IRESs of the Dicistroviridae, which drive a highly streamlined mode of ribosome recruitment and activation. Over the last few years, we have made discoveries that lead to a detailed structure-based model of IGR IRES function; we are now poised to test that model and explain the function of an IRES in unprecedented detail. We propose to do this with three specific aims. First, we will employ single molecule FRET (smFRET) to observe the motions of IRES RNAs, tRNAs, and ribosome components within an initiating complex. By watching how the structured IRES drives specific movements on the ribosome and comparing these motions to those of canonical elongation, we will gain insight at a level not yet achieved. Second, we will explore conformational changes within the IRES RNA structure itself, linking these dynamic changes to global changes in the initiating complex and in so doing, turn static pictures into dynamic pathways. Third, we will solve the structures of IRES-ribosome complexes by x-ray crystallography, which should reveal heretofore unseen intimate ribosome-IRES interactions that underlie conformational changes. These three independent but complementary aims promise to yield a cohesive, high-resolution, and dynamic mechanistic picture of how an IRES RNA manipulates a fundamental biological machine, lending wide-ranging insight into universal biological processes.