Translational Fidelity in Eukaryotes

摘要 随着核糖体原子级结构的出现，下一个关键任务是将核糖体连接起来 具有生物功能的结构。在生物学层面上，我们一直在探索核糖体如何识别 使用病毒的翻译重新编码信号终止密码子和维持翻译阅读框架 起源。病毒重新编码对病毒的传播很重要，它们基于mRNA的重新编码信号具有 事实证明，它在阐明这些基本任务背后的分子机制方面非常有用。 这些研究都是基于酵母酿酒酵母模型真核生物 因为它为研究人员提供了最先进、最多样化和最强大的工具箱。此外， 我们已经在这个基础上开发了一个强大的和协同的分子遗传组合， 生化、结构和分子建模工具。这使我们能够表明， 核糖体蛋白、rRNAs和tRNAs之间的生物物理相互作用及其生化性质 核糖体相关的酶活性对于正确的阅读框架的维持和停止是重要的 密码子识别。在更广泛的范围内，我们的工作是定义变构通信路径， 核糖体的不同功能中心相互连接和协调。的宏伟目标 这项提议是为了进一步定义核糖体结构如何影响功能。目标1将决定 靶向突变对酵母核糖体结构和功能的影响。具体地说，反向遗传学 将应用各种方法来确定特定核糖体蛋白和核糖体RNA的功能。 这些研究包括扩展以检查两个与核糖体相关的突变对 哺乳动物的系统。第二个目标将描述核糖体之间的相互作用 麻痹病毒内部核糖体进入信号(CRPV IRES)与HIV-1程序化核糖体 移码信号。提议的协作代表了我们的工作在逻辑上扩展到新和 激动人心的领域。我们还预计，在拟议的研究过程中，将取得突破 在核糖体结构领域继续取得进展，与核糖体和核糖体相关的新发现 疾病将会揭开面纱。拟议的计划将使我们能够迅速利用这些优势，提供 为新的和意想不到的发现机会奠定坚实的基础。最终，这项工作将使 对科学界和临床界的重大贡献通过加深我们对 核糖体结构和功能之间的关系，同时拓宽了我们对翻译保真度的看法 和疾病。项目叙事 增殖的细胞，无论是胚胎细胞忙于创造新人，还是T细胞在战斗 脱离感染，或癌细胞压倒患者，绝对需要大量的 高度精确的核糖体，以满足它们合成新蛋白质的需要。 核糖体是这一过程的中心组成部分，是复杂的生物学过程。 由许多蛋白质和RNA分子组成的纳米机器，其总体目标是 拟议的研究是为了开始了解原子尺度结构是如何 核糖体最终决定其功能。加深对两国关系的理解 核糖体结构和功能之间的关系将有助于合理设计新的核糖体 针对多种临床应用而设计的药物，包括抗病毒和 抗菌剂，以及针对多种癌症的药物， 发育障碍，以及其他困扰社会的危重疾病。

ABSTRACT With the availability of atomic scale structures of ribosomes, the next critical task is to link ribosome structure with biological function. At the biological level, we have been exploring how ribosomes recognize termination codons and maintain translational reading frame using translational recoding signals of viral origin. Viral recoding is important for virus propagation, and their mRNA-based recoding signals have proved to be of great utility in elucidating the molecular mechanisms underlying these essential tasks. These studies have been based on the yeast Saccharomyces cerevisiae model eukaryotic organism because it provides researchers with the most advanced, diverse, and robust toolbox available. Further, we have built upon this foundation to develop a robust and synergistic combination of molecular genetic, biochemical, structural, and molecular modeling tools. This has enabled us to show that both the biophysical interactions between ribosomal proteins, rRNAs and tRNAs, and the biochemical properties of ribosome-associated enzymatic activities are important for proper reading frame maintenance and stop codon recognition. On a broader scale, our work is defining the allosteric communication pathways that connect and coordinate different functional centers of the ribosome with one another. The broad goal of this proposal is to further define how ribosome structure influences function. Aim 1 will determine the effects of targeted mutations on yeast ribosome structure and function. Specifically, reverse genetics approaches will be applied to define the functions of specific ribosomal proteins and ribosomal RNAs. These studies include expansion to examine the effects of two ribosome-associated mutations in mammalian systems. The second aim will characterize the interactions between ribosomes the Cricket Paralysis Virus Internal Ribosome Entry Signal (CRPV IRES), and the HIV-1 programmed -1 ribosomal frameshift signal. The proposed collaborations represent logical expansions of our work into new and exciting areas. We also anticipate that during the course of the proposed studies, breakthroughs will continue to be made in the area of ribosome structure, and that new discoveries relevant to ribosomes and disease will be unveiled. The proposed program will position us to quickly capitalize on these, providing a strong foundation for new and unanticipated discovery opportunities. In the end, this work will make significant contributions to the scientific and clinical communities by both deepening our understanding of the relationship between ribosome structure and function, while broadening our view of translational fidelity and disease. PROJECT NARRATIVE Proliferating cells, be they embryonic cells busily creating new persons, T-cells fighting off infection, or cancer cells overwhelming the patient, absolutely require large numbers of highly accurate ribosomes to meet their needs for synthesis of new proteins. Ribosomes, the central component of this process, are complex biological nanomachines composed of many protein and RNA molecules, and the overall goal of the proposed research is to begin to understand how the atomic scale structure of the ribosome ultimately determines its function. A deeper understanding of the relationship between ribosome structure and function will aid the rational design of new classes of drugs designed to target a diverse array of clinical applications including antiviral and antibacterial agents, as well as drugs targeting a diverse array of cancers, developmental disorders, and other critical diseases afflicting society.