BIOSYNTHESIS OF RNAS

RNAS的生物合成

• 批准号: 2173651
• 负责人: CHRISTINE GUTHRIE
• 金额: $ 52.12万
• 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 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2173651
• 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