BIOSYNTHESIS OF RNAS

RNAS的生物合成

• 批准号: 2872641
• 负责人: CHRISTINE GUTHRIE
• 金额: $ 60.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-02-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872641
• 关键词: RNA biosynthesis; RNA splicing; Saccharomyces cerevisiae; adenosine triphosphate; alleles; conformation; evolution; fungal genetics; genetic transcription; guanine nucleotide binding protein; immunochemistry; lethal genes; messenger RNA; molecular cloning; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis; small nuclear ribonucleoproteins; spliceosomes; temperature sensitive mutant

Our long-term objectives are to exploit the powerful techniques available in the yeast Saccharymocyes cerevisiae for an understanding of messenger RNA splicing at the molecular level. Our future work relies on our recent demonstration of a 1:1 correspondence between five small nuclear RNAs (snRNAs) in this yeast and mammalian structural organization. One focus of your program -- motivated by the discovery of group II self-splicing RNAs - - will be to seek catalytic roles for these snRNAs. We will identify those residues that are the best candidates for such functions using a combined genetic, phylogenetic, and biochemical approach. First, structural domains inferred from phylogenetic comparisons will be deleted and assayed by complementation of deletion strains. When an essential domain is identified, its function will be further assessed by inter-species "swaps"; this provides a rapid and rational method of bulk mutagenesis. Finally, evolutionarily invariant nucleotides in these chimeras will by subjected to site-specific mutagenesis, to identify change which lead to lethal or, ideally, conditionally lethal phenotypes. To determine the specific functional lesion, we will assay the pattern of splicing intermediates in vivo, and the distribution of spliceosomal complexes within the ordered assembly pathway in vitro. The complementary focus of this project is to understand what roles the spliceosomal proteins play, many of which are known to be essential for viability. We will explore the hypothesis that at least certain of these proteins participate in proofreading functions, consistent with the large number of steps in the spliceosome assembly pathway that require ATP. In particular, we have cloned and sequenced a nucleotide; rna16-1 has a consensus ATP binding site and other features of a recently reported superfamily, members of which include EIF4alpha and a protein required for mitochondrial mRNA splicing. We propose genetic and biochemical tests of the model that RNA16 functions in branchpoint recognition to effect an ATP- dependent conformational switch; by this view, rna16-1 is a "clock mutant" that acts as a suppressor by decreasing the time allowed for incorrect splicing substrates to dissociate. This model has important consequences for elucidating the molecular mechanisms used to maintain biologically tolerable error rates in complex macromolecular precesses.

我们的长期目标是利用现有的强大技术 在酿酒酵母中， 分子水平上的RNA剪接。 我们未来的工作依赖于我们最近的 证明了5个小核RNA之间的1：1对应关系 （snRNAs）在这种酵母和哺乳动物的结构组织。 的一个重点 你的项目--由第二组自我剪接RNA的发现激发-- - 将是寻找这些snRNA的催化作用。 我们会找出那些 残基是这样的功能的最佳候选人，使用组合 遗传、系统发育和生化方法。 第一，结构域 从系统发育比较中推断的基因将被删除，并通过 缺失菌株的互补。 当一个基本域是 将通过物种间“互换”进一步评估其功能; 这提供了一种快速和合理的批量诱变方法。 最后， 这些嵌合体中进化上不变的核苷酸将受到 位点特异性诱变，以鉴定导致致死或， 理想情况下是条件致死表型。 确定具体 功能性损伤，我们将分析剪接中间体的模式， 体内，以及剪接体复合物在有序细胞内的分布。 体外组装途径。 该项目的补充重点是了解 剪接体蛋白发挥作用，其中许多已知是必不可少的 生存能力。 我们将探讨的假设，至少某些这些 蛋白质参与校对功能，与大的 剪接体组装途径中需要ATP的步骤数量。 在 特别是，我们克隆并测序了一个核苷酸; rna 16 -1具有一个 一致的ATP结合位点和最近报道的 超家族，其成员包括EIF 4 α和EIF 4 α所需的蛋白质。 线粒体mRNA剪接。 我们建议进行基因和生化测试， RNA 16在分支点识别中起作用以影响ATP- 依赖性构象转换;根据这种观点，rna 16 -1是一种“时钟突变体” 作为一个抑制器，通过减少时间允许不正确的 拼接底物以解离。 这种模式具有重要的后果 用于阐明用于生物维持的分子机制 在复杂的大分子过程中可容忍的错误率。