Biochemical and Genetic Analysis of the RNA Polymerase Specificity of Small Nuclear RNA Genes

小核 RNA 基因的 RNA 聚合酶特异性的生化和遗传分析

• 批准号: 9818000
• 负责人: William Stumph
• 金额: $ 33万
• 依托单位: San Diego State University Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9818000&HistoricalAwards=false
• 关键词: Biochemical; Genetic; Analysis; RNA; Polymerase

Stumph The small nuclear RNAs (snRNAs) known as U1, U2, U4, U5, and U6 comprise a highly abundant class of metabolically stable, non-polyadenylated RNA molecules that are required for pre-messenger RNA splicing in eukaryotic organisms. Whereas U6 snRNA is synthesized by RNA polymerase III, the remainder of the spliceosomal snRNAs are synthesized by RNA polymerase II. In spite of this difference in polymerase specificity, the promoter sequences of U6 genes are very similar in structure to those of the snRNA genes transcribed by RNA polymerase II. For example, U1 and U6 genes have promoter structures that are more closely related to each other than either is related to classical RNA polymerase II or III promoters. Furthermore, overlapping sets of transcription factors participate in binding to U1 and U6 promoters, including the TATA-binding protein (TBP) and the PSE-binding protein (PBP). It is therefore of great interest to understand how RNA polymerase specificity is determined at snRNA gene promoters. Experiments will be performed that use the U1 and U6 snRNA genes of the fruit fly Drosophila melanogaster as a model system. A multifaceted approach using techniques of biochemistry, molecular biology, molecular modeling, and genetics will be employed. The trajectory of the DNA will be determined following its assembly into partial transcription pre-initiation complexes with PBP and TBP. Helical phasing, DNA circularization, and DNA minicircle binding assays will be used to determine the DNA-bending properties of PBP itself bound to DNA. Following this, the overall net direction of DNA bending will be determined when PBP and TBP are simultaneously bound to the same DNA molecule. The unique features of the Drosophila system will be further exploited by employing a genetic approach. A screen for Drosophila mutants that have an altered or relaxed RNA polymerase specificity at snRNA gene promoters will be carried out. The defective genes will be mapped, isolated, and characterized. There are two classes of enzymes that synthesize the small nuclear RNAs (snRNAs) that function in mRNA maturation: RNA polymerase II and RNA polymerase III. The regulatory DNA sequences that specify which polymerase is used for a given snRNA are quite similar, so it is of interest to determine what specifies the choice. In this project, the organization of the proteins that initiate snRNA synthesis will be examined in RNA pol II and RNA pol III-specific snRNA genes, using biochemical approaches. Furthermore, Drosophila mutants that can use either RNA pol II or RNA pol III will be isolated and characterized. This work should lead to significant insight into the molecular architecture that effects the choice of RNA polymerase not only on snRNA promoters, but also on other promoters transcribed by RNA polymerases II and III.

小核RNA （snrna）被称为U1、U2、U4、U5和U6，包括一类代谢稳定的、非聚腺苷化的RNA分子，它们是真核生物中前信使RNA剪接所必需的。而U6 snRNA是由RNA聚合酶III合成的，其余的剪接体snRNA是由RNA聚合酶II合成的。尽管在聚合酶特异性上存在差异，但U6基因的启动子序列在结构上与RNA聚合酶II转录的snRNA基因非常相似。例如，U1和U6基因的启动子结构与经典RNA聚合酶II或III启动子的关系更为密切。此外，重叠的转录因子组参与与U1和U6启动子的结合，包括tata结合蛋白（TBP）和pse结合蛋白（PBP）。因此，了解RNA聚合酶特异性是如何在snRNA基因启动子上确定的是非常有趣的。实验将以果蝇（Drosophila melanogaster）的U1和U6 snRNA基因为模型系统。使用生物化学、分子生物学、分子建模和遗传学技术的多方面方法将被采用。DNA的轨迹将在其与PBP和TBP组装成部分转录起始前复合物后确定。螺旋相位，DNA环状化和DNA微环结合分析将用于确定PBP本身与DNA结合的DNA弯曲特性。随后，当PBP和TBP同时结合到同一DNA分子上时，将确定DNA弯曲的总体净方向。果蝇系统的独特特征将通过采用遗传方法进一步开发。筛选在snRNA基因启动子上具有改变或放松RNA聚合酶特异性的果蝇突变体。缺陷基因将被定位、分离和表征。有两类酶可以合成在mRNA成熟过程中起作用的小核RNA (snrna): RNA聚合酶II和RNA聚合酶III。指定用于特定snRNA的聚合酶的调控DNA序列非常相似，因此确定是什么指定了选择是很有趣的。在本项目中，将使用生化方法研究RNA pol II和RNA pol iii特异性snRNA基因中启动snRNA合成的蛋白质组织。此外，可以使用RNA pol II或RNA pol III的果蝇突变体将被分离和表征。这项工作将有助于深入了解影响RNA聚合酶选择的分子结构，这些结构不仅影响snRNA启动子，还影响RNA聚合酶II和III转录的其他启动子。