Description
In Drosophila melanogaster and other higher eukaryotes there are five major spliceosomal small nuclear RNAs (snRNAs): U1, U2, U4, U5 and U6. These non-coding snRNAs are important in RNA processing events such as pre-mRNA splicing. snRNA gene transcription is dependent upon two conserved promoter sequences, approximately 25 to 60 base pairs (bps) upstream of the transcription start site, which are essential for proper RNA polymerase recruitment. U1, U2, U4, and U5 snRNA genes contain a 21 bp Proximal Sequence Element A (PSEA) located 8 bp upstream from an 8 bp Proximal Sequence Element B (PSEB), and recruit RNA Polymerase II for transcription. Unlike those genes, U6 gene promoters contain an 8 bp TATA box, instead of a PSEB, located 12 bp downstream of the U6 PSEA, and recruit RNA polymerase III. Despite similarities in length and distance from the transcription start site, U1 and U6 PSEAs are non-interchangeable. U1 and U6 PSEAs are specifically recognized and bound by a multi-subunit transcription factor, the D. melanogaster snRNA-activating protein complex (DmSNAPc). DmSNAPc consists of three subunits: DmSNAP190, DmSNAP50, and DmSNAP43, all of which are orthologous to those of human SNAPc. Previous studies, utilizing site-specific protein-DNA photo-cross-linking assays, showed that upon interaction with a U6 PSEA, subunits of DmSNAPc adopt a conformation unique from that formed upon its interaction with a U1 gene PSEA. This thesis focuses on DmSNAP190, the only DmSNAPc subunit that contains a canonical DNA-binding domain. This domain is comprised of 4.5 tandem Myb repeats, first identified in the Myb oncoprotein's DNA-binding domain. I was able to localize regions within DmSNAP190 that interact with specific U6 PSEA phosphate positions by utilizing a novel technique developed in our lab: site-specific protein-DNA photo-cross-linking coupled with site-specific hydroxylamine digestion. My studies show that positions at the 5' end of the U6 PSEA cross-link with the Myb repeats. Surprisingly, however, the DmSNAP190-DNA interactions are not limited to DmSNAP190's Myb domain. In fact, the region N-terminal of DmSNAP190's Myb domain is in close proximity to the 3' half of the U6 PSEA.
