BIOSYNTHESIS OF RNAS

RNAS的生物合成

• 批准号: 2173650
• 负责人: CHRISTINE GUTHRIE
• 金额: $ 52.22万
• 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 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173650
• 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 酿酒酵母中的信使 分子水平上的RNA剪接。 我们未来的工作依赖于我们最近的工作 演示五个小核 RNA 之间的 1:1 对应关系 (snRNA) 在这种酵母和哺乳动物的结构组织中。 重点之一 你的计划——受到 II 族自剪接 RNA 的发现的启发—— - 将寻找这些 snRNA 的催化作用。 我们将识别那些 使用组合的残基是此类功能的最佳候选残基 遗传学、系统发育和生化方法。 一、结构域 从系统发育比较推断的将被删除并通过 缺失菌株的互补。 当基本域是 确定后，将通过物种间“互换”进一步评估其功能； 这提供了一种快速且合理的批量诱变方法。 最后， 这些嵌合体中进化不变的核苷酸将受到 位点特异性诱变，识别导致致命的变化，或者， 理想情况下，有条件致死的表型。 来确定具体 功能性损伤，我们将检测剪接中间体的模式 体内，以及剪接体复合物在有序内的分布 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，其中许多已知对于 生存能力。 我们将探讨以下假设：至少其中某些 蛋白质参与校对功能，与大的一致 剪接体组装途径中需要 ATP 的步骤数。 在 特别是，我们已经克隆并测序了一个核苷酸； rna16-1 有一个 共有 ATP 结合位点和最近报道的其他特征 超家族，其成员包括 EIF4alpha 和 线粒体 mRNA 剪接。 我们建议进行基因和生化测试 RNA16 在分支点识别中发挥作用以影响 ATP 的模型 依赖构象开关；由此看来，rna16-1是一个“时钟突变体” 通过减少错误允许的时间来充当抑制器 拼接底物以解离。 该模型具有重要的影响 阐明用于维持生物学状态的分子机制 复杂大分子过程中可容忍的错误率。