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.
