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

Stumph小核RNA(SnRNAs)，即U1、U2、U4、U5和U6，是真核生物中前信使RNA剪接所必需的一类高度丰富的代谢稳定的非多腺化RNA分子。尽管U6基因的启动子序列与RNA聚合酶II转录的SnRNA基因序列在结构上非常相似，但U1和U6基因的启动子结构比经典的RNA聚合酶II或传统的RNA聚合酶II或III启动子的结构更密切。此外，重叠的转录因子参与与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聚合酶的特异性是如何在SnRNA基因启动子上决定的是非常有意义的。将以果蝇U1和U6的SNRNA基因作为模型系统进行实验。将使用生物化学、分子生物学、分子建模和遗传学等技术的多方面方法。DNA的轨迹将在它与PBP和TBP组装成部分转录预启动复合体后确定。螺旋相变、DNA环化和DNA小环结合试验将被用来确定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，DNA弯曲的总体净方向将被确定。通过采用遗传学方法，将进一步开发果蝇系统的独特功能。将进行对SnRNA基因启动子具有改变或放松的RNA聚合酶专一性的果蝇突变体的筛选。有缺陷的基因将被定位、分离和鉴定。有两类酶可以合成在mRNA成熟过程中起作用的小核RNA(SnRNAs)：RNA聚合酶II和RNA聚合酶III。指定哪个聚合酶用于给定的SnRNA的调控DNA序列非常相似，因此确定是什么指定了选择是有意义的。在这个项目中，将使用生化方法在RNAPolII和RNAPolIII特定的SnRNA基因中检查启动SnRNA合成的蛋白质的组织。此外，还将分离和鉴定可以使用RNAPolII或RNAPolIII的果蝇突变体。这项工作应该导致对影响RNA聚合酶的选择的分子结构的重大洞察，该分子结构不仅影响SnRNA启动子，而且影响由RNA聚合酶II和III转录的其他启动子。

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.