BIOSYNTHESIS OF RNAS

我们的长期目标是利用现有的强大技术 在酵母中了解酿酒酵母中的信使 RNA在分子水平上的剪接。我们未来的工作依赖于我们最近的 5个小核RNA之间1：1对应的演示 (SNRNAs)在这种酵母和哺乳动物的结构组织中。关注的焦点之一 你们的计划--以发现第二组自剪接RNA为动力-- -将为这些SnRNA寻求催化作用。我们会找出那些 残基是此类函数的最佳候选者，使用组合的 遗传、系统发育和生化方法。第一，结构性领域 从系统发育比对中推断出来的将被删除并由 缺失菌株的互补性。当一个必要的域是 一旦确定，将通过物种间“互换”进一步评估其功能； 这为大规模诱变提供了一种快速、合理的方法。最后， 这些嵌合体中进化上不变的核苷酸将受到 定点突变，以确定导致致命或， 理想情况下，有条件致死的表型。要确定特定的 功能损伤，我们将分析剪接中间产物在 Vivo，以及剪接体复合体在体内的有序分布 体外组装途径。 本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。我们将探索这样一种假设，即至少其中某些 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。在……里面 特别是，我们已经克隆并测序了一个核苷酸；rna 16-1具有一个 最近报道的一种共识三磷酸腺苷结合位点等特征 超家族，其成员包括EIF4α和一种蛋白质 线粒体mRNAs拼接。我们建议进行遗传和生化测试 RNA16在分支点识别中作用的模型影响ATP- 依赖的构象开关；从这个观点来看，rna 16-1是一个“时钟突变体” 它通过减少错误允许的时间来起到抑制的作用 拼接底物以解离。这种模式具有重要的后果。 为了阐明用于维持生物学的分子机制 复杂大分子过程中可容忍的错误率。

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.