Translation Initiation Factor IF3 Structure and Function

摘要MCB-0079305SimonsSIMONST核糖体是一个复杂的分子机器，它将储存在遗传密码中的信息忠实地翻译成酶和其他蛋白质。了解核糖体的功能一直是分子生物学中最重要的挑战之一。这个项目的长期目标是了解核糖体如何确保其P位点的翻译准确性(正确解码)。P-位点在翻译起始过程中对信息进行解码，20多年的研究表明，翻译起始因子3(IF3)在这一事件中发挥着关键作用，几乎可以肯定的是，它通过调节核糖体本身固有的活动来发挥作用。在过去的5年里，出现了重要的新见解，包括IF3和核糖体的三维结构，IF3/核糖体复合体的可视化，以及对IF3结构和功能的新见解。IF3被认为作用于或接近P-位点的功能水平，极有可能改变核糖体的构象，从而改变P-位点。这种对P位点活性的调节可能表现在几个看似不相关的IF3效应中。这些发展为最终理解P-位点的功能以及IF3对其的调控带来了巨大的希望。然而，在合理地实现这些重要目标之前，必须首先解决IF3结构和功能的几个基本和关键方面。这个项目涉及到在这个层面上对几个重大悬而未决的问题进行综合的遗传和生化攻击。关键的IF3突变的影响将通过一系列强大的分析来检验，这些分析旨在监测IF3在几个不同的核糖体功能水平上的功能。这些努力有助于测试核糖体功能的IF3调节的现有模型。还将探索与核糖体相互作用所需的IF3决定因素，并将根据现有实验证据确定核糖体本身内的IF3结合决定因素。这些研究对于最终了解IF3的结构和功能如何与核糖体整合至关重要。特别是，这项工作将为最终直接在P-现场解决IF3功能的研究奠定坚实的基础。

AbstractMCB-0079305SimonsSIMONSThe ribosome is a complex molecular machine that faithfully translates information stored in the genetic code, into enzymes and other proteins. Understanding ribosome function has been and remains one of the most important challenges in molecular biology. The long-term goal of this project is to understand how the ribosome ensures translational accuracy (proper decoding) at its P-site. The P-site decodes information during translation initiation, and work spanning more that two decades has shown that translation initiation factor three (IF3) plays a crucial role in this event, almost certainly by modulating activities that are intrinsic to the ribosome itself. During the past 5 years, important new insights have emerged, including the three-dimensional structure of IF3 and the ribosome, visualization of the IF3/ribosome complex, and new insights into IF3 structure and function. IF3 is thought to operate at or close to the level of P-site function, most likely altering ribosome conformation and thereby the P-site. This modulation of P-site activity probably manifests itself in several seemingly unrelated IF3 effects. These developments hold great promise for an eventual understanding of P-site function and its modulation by IF3. However, before those important goals can be reasonably tackled, several basic and key aspects of IF3 structure and function must first be resolved. This project involves an integrated genetic and biochemical attack on several major unanswered questions at this level. The effects of key IF3 mutations will be examined with a powerful battery of assays designed to monitor IF3 function at several distinct levels of ribosome function. These efforts are instrumental in testing current models for IF3 modulation of ribosome function. IF3 determinants required for interaction with the ribosome will also be explored, and the IF3 binding determinants within the ribosome itself will be identified, in strategies based on existing experimental evidence. These studies are essential for an eventual understanding of how IF3 structure and function are integrated with that of the ribosome. In particular, this work will form a strong underpinning for studies that will eventually tackle IF3 function directly at the P-site.