UC San Diego

In animals, the U1, U2, U4 and U5 small nuclear RNA (snRNA) genes are transcribed by RNA polymerase (RNAP) II, but U6 snRNA genes are transcribed by RNAP III. Transcription of both classes of genes is dependent upon a 21 base pair (bp) sequence termed the PSEA located ̃40- 60bp upstream of the transcription start site. Other promoter elements consist of a TATA box (in U6) and a PSEB (in U1-U5). The PSEAs of both classes of Drosophila snRNA genes are recognized by the same transcription factor, DmSNAPc (Drosophila melanogaster snRNA activating protein complex), which comprises three distinct subunits (DmSNAP43, DmSNAP50 and DmSNAP190). A striking previous finding was that the DmSNAP43 subunit cross-links to DNA more than 20 bp downstream of the U1 PSEA (a region that includes the PSEB). These findings raise the question of whether the PSEB contributes to the cross-linking pattern downstream of the U1 PSEA. To investigate this, the photo- cross-linking patterns from wild type or mutant PSEB probes were compared. Both sets of probes produced a similar, although not identical, photo-cross-linking pattern. These results indicate that the PSEA itself can bring DmSNAP43 into close proximity to the downstream DNA regardless of the PSEB sequence. A second part of this study focused on the stoichiometry of the subunits of DmSNAPc bound to DNA. To investigate this, identical subunits were tagged with different epitopes and co- expressed in Drosophila S2 cells with each other and the other two subunits. Following purification of the tagged DmSNAPc the presence of differently tagged subunits in DmSNAPc bound to DNA was investigated by band-shift and super-shift assays. The results indicate that each of the subunits is present in only a single copy in DmSNAPc bound to DNA. A third part of this study focused on the N- and C -terminal orientation of the largest subunit, DmSNAP190, when bound to the U1 promoter. By combining the photo- cross-linking assay with chemical digestion of the protein, I have been able to demonstrate that the N-terminal half of DmSNAP190 contacts the 3' end of PSEA and most likely the C-terminal half contacts the 5' end of the PSEA