RIBOSOMAL FRAMESHIFTING AS A PROBE OF 5S RRNA FUNCTION

核糖体移码作为 5S RRNA 功能的探针

• 批准号: 6558675
• 负责人: Jonathan D Dinman
• 金额: $ 2.71万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6558675
• 关键词: aminoacid tRNA ligase; chemical structure function; enzyme activity; frameshift mutation; genetic translation; guanosinetriphosphatases; intermolecular interaction; oligonucleotides; protein biosynthesis; ribosomal RNA; ribosomal proteins; site directed mutagenesis

DESCRIPTION (Applicant's abstract): The ribosome is the central component of an extremely accurate cellular protein synthetic apparatus. Its job is to rapidly and accurately decode mRNAs by reading three base "codons." Although it is exquisitely designed to maintain translational reading frame, there are several naturally occurring instances in which this process has been subverted, one of which is called programmed ribosomal frameshifting (PRF). Such exceptions to the rule provide a powerful set of tools to elucidate the molecular mechanisms underlying the normal maintenance of translational reading frame. With the advent of molecular genetics, it has been possible to create and examine the effects of mutants of individual ribosomal components on different ribosome-associated functions using specialized assay systems. The application of these new tools to classic biochemical methods are leading to a deeper understanding of the roles that many of the ribosomal proteins and ribosomal RNAs (rRNAs) play in determining how ribosomes maintain translational reading frame. It is now clear that the rRNAs are the central players in the reactions catalyzed by ribosomes, and that the individual rRNAs are actively involved in different ribosome functions. However, although it is highly conserved throughout evolution, the precise function of the ubiquitous 5S rRNA remains undetermined. Our demonstration that mutants of the yeast 5S rRNA (called mof9) can alter PRF efficiencies provides us with a unique platform from which investigations into the function of 5S rRNA can be launched. Preliminary results are consistent with recent structural reports suggesting a role for 5S rRNA peptidyltransferase center and A-site associated functions. The studies proposed herein will use PRF as a primary indicator of function to genetically and biochemically characterize 5S rRNA. Two complementary yeast systems will be utilized to monitor the affects of specific 5S rRNA mutants on PRF efficiencies, nonsense suppression, and cell growth and viability. Biochemical studies will be performed to test for specific functional and structural defects due to the mof9 mutants. Moreover, given the demonstrated physical interactions between 5S rRNA and ribosomal protein L5 (L5), we will follow up on preliminary studies to determine how this protein also contributes to translational reading frame maintenance. The proposed research will serve to further our understanding of this ubiquitous yet little understood rRNA and will place us in the unique position to link functional aspects of 5S rRNA to its structure within the ribosome.

描述（申请人摘要）：核糖体是核糖体的核心成分。 极其精确的细胞蛋白质合成装置。它的工作是迅速 并通过阅读三个碱基的密码子来精确地解码mRNA。“虽然它是 精心设计，以保持翻译阅读框架，有几个 自然发生的情况下，这一进程已被颠覆，其中之一， 这被称为程序性核糖体移码（PRF）。此类例外情况 该规则提供了一套强有力的工具来阐明分子机制 翻译阅读框架的正常维持。与 随着分子遗传学的出现，已经有可能创造和检查 单个核糖体组分的突变体对不同的 核糖体相关的功能，使用专门的分析系统。应用 这些新的工具，经典的生化方法正在导致更深层次的 了解许多核糖体蛋白和核糖体蛋白的作用， 核糖核酸（rRNA）决定核糖体如何维持翻译阅读 frame.现在很清楚，rRNAs是反应的核心参与者 由核糖体催化，并且单个rRNA积极参与 不同的核糖体功能。然而，尽管它是高度保守的， 在整个进化过程中，普遍存在的5S rRNA的精确功能仍然存在， 不确定。我们证明酵母5S rRNA突变体（称为mof9） 可以改变PRF效率为我们提供了一个独特的平台， 可以对5S rRNA的功能进行研究。初步 结果与最近的结构报告一致，表明5S的作用 rRNA肽基转移酶中心和A位点相关功能。研究 本文提出的将使用PRF作为功能的主要指标， 并对5S rRNA进行生物化学表征。两个互补的酵母系统将 用于监测特定5S rRNA突变体对PRF的影响 效率、无义抑制以及细胞生长和活力。生化 将进行研究，以测试特定的功能和结构 mof9突变体导致的缺陷。此外，鉴于所展示的物理 5SrRNA和核糖体蛋白L5（L5）之间的相互作用，我们将跟踪 进行初步研究，以确定这种蛋白质如何有助于 翻译阅读框架维护。拟议的研究将有助于 进一步加深了我们对这种普遍存在但知之甚少的rRNA的理解， 将使我们处于独特的位置，将5S rRNA的功能方面与 它在核糖体中的结构。