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.

stumph被称为U1，U2，U4，U5和U6的小核RNA（SNRNA）组成了一类高度丰富的代谢稳定，非多丙基烯基化的RNA分子，这些分子在真核生物中所需的预兆剂RNA所需的RNA所需。 而u6 snRNA由RNA聚合酶III合成，其余的剪接体SNRNA由RNA聚合酶II合成。 尽管在聚合酶特异性方面存在这种差异，但U6基因的启动子序列与RNA聚合酶II转录的SnRNA基因的结构非常相似。 例如，U1和U6基因的启动子结构与彼此相关的启动子结构比与经典RNA聚合酶II或III启动子有关的启动子结构。 此外，重叠的转录因子集参与与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，了解如何在SnRNA基因启动子上确定RNA聚合酶特异性是很大程度上的。 将进行实验，以使用果蝇果蝇的U1和U6 SnRNA基因作为模型系统。 将采用生物化学，分子生物学，分子建模和遗传学技术的多方面方法。 DNA的轨迹将在将其组装成与PBP和TBP的部分转录前发射复合物中确定。 螺旋相位，DNA圆形和DNA微圆界结合测定将用于确定与DNA结合的PBP本身的DNA弯曲特性。 此后，当PBP和TBP同时结合到同一DNA分子时，将确定DNA弯曲的总净方向。 果蝇系统的独特特征将通过采用遗传方法进一步利用。 将进行果蝇突变体的屏幕，该突变体将在SnRNA基因启动子上具有改变或松弛的RNA聚合酶特异性。 有缺陷的基因将被映射，隔离和表征。 有两类的酶合成在mRNA成熟中起作用的小核RNA（SNRNA）：RNA聚合酶II和RNA聚合酶III。指定哪种聚合酶用于给定SNRNA的调节DNA序列非常相似，因此确定指定选择的是很相似。在这个项目中，将使用生化方法在RNA POL II和RNA POL III特异性SNRNA基因中检查启动SNRNA合成的蛋白质的组织。此外，将分离和表征可以使用RNA POL II或RNA POL III的果蝇突变体。这项工作应导致对分子结构的重大见解，该分子结构不仅在SNRNA启动子上影响RNA聚合酶的选择，而且还影响了RNA聚合酶II和III转录的其他启动子。