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可用的强大技术 在酵母saccharymocyes cerevisiae中，以了解使者 RNA剪接在分子水平上。 我们未来的工作依赖于我们最近的 五个小核RNA之间的1：1对应关系的证明 （SNRNA）在这个酵母和哺乳动物结构组织中。 一个焦点 您的计划 - 发现II组自剪接RNA的动机 - - 将为这些SNRNA寻求催化作用。 我们将确定那些 残留物是使用合并的最佳候选者 遗传，系统发育和生化方法。 首先，结构域 从系统发育比较推断的将被删除和测定 缺失菌株的补充。 当必需域是 确定，其功能将通过种间“掉期”进一步评估。 这提供了散装诱变的快速而合理的方法。 最后， 这些嵌合体中进化的不变核苷酸将受到 位点特异性诱变，以确定导致致命或，， 理想情况下，有条件致命的表型。 确定特定 功能性病变，我们将分析剪接中间体的模式 体内和剪接体复合物的分布 体外组装途径。 该项目的互补重点是了解角色 剪接体蛋白可以玩，其中许多是必不可少的 生存能力。 我们将探讨至少某些这些假设 蛋白质参与校对功能，与大型 需要ATP的剪接组装路径中的步骤数。 在 特别是，我们已经克隆并测序了核苷酸。 RNA16-1有一个 共识ATP绑定站点和最近报道的其他功能 超家族，其中的成员包括EIF4Alpha和所需的蛋白质 线粒体mRNA剪接。 我们提出了遗传和生化测试的 RNA16在分支点识别中起作用以实现ATP-的模型 依赖构象开关；通过这种观点，RNA16-1是“时钟突变体” 通过减少允许的时间不正确的时间来起到抑制作用 剪接基板以解离。 该模型有重要的后果 用于阐明用于维持生物学的分子机制 复杂的大分子进攻中的可耐受性错误率。