RIBOSOMAL FRAMESHIFTING AS A PROBE OF 5S RRNA FUNCTION

描述(申请人摘要)：核糖体是核糖体的中心成分。 极其精确的细胞蛋白质合成设备。它的工作是迅速 并通过阅读三个碱基“密码子”来准确地解码mRNA。尽管它是 精心设计，以保持翻译阅读框架，有几个 这一过程被颠覆的自然发生的情况之一， 这被称为程序性核糖体移码(PRF)。此类例外情况 该规则为阐明分子机制提供了一套强大的工具 翻译阅读框架的正常维护。与 分子遗传学的出现，已经有可能创造和研究 单个核糖体成分突变对不同核糖体的影响 使用专门的检测系统实现核糖体相关功能。应用程序 这些新工具中的经典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的作用 RNA(RRNA)在决定核糖体如何维持翻译阅读中发挥作用 框架。现在很明显，rRNA是反应中的中心角色。 由核糖体催化，单个rRNA积极参与 不同的核糖体功能。然而，尽管它高度保守， 在整个进化过程中，无处不在的5S rRNA的确切功能仍然存在 未定。我们的研究表明酵母5S rRNA(称为mof9)的突变体 可以改变PRF效率为我们提供了一个独特的平台 可以启动对5S rRNA功能的调查。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。这些研究 这里提出的将使用PRF作为遗传功能的主要指标 并对5S rRNA进行了生化表征。两个互补的酵母系统将是 用于监测特定的5S rRNA突变体对PRF的影响 效率，无意义的抑制，以及细胞的生长和存活。生化 将进行研究，以测试特定的功能和结构 由mof9突变体引起的缺陷。此外，考虑到所展示的身体状况 5S rRNA与核糖体蛋白L5(L5)的相互作用，我们将继续跟踪 关于初步研究，以确定这种蛋白质如何也有助于 翻译阅读框维护。拟议的研究将有助于 进一步加深我们对这种普遍存在但知之甚少的rRNA和 将使我们处于将5S rRNA的功能方面与 它在核糖体内的结构。

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.