UC San Diego

The small nuclear RNA activating protein complex (SNAPc) is necessary for transcription of genes coding for the spliceosomal snRNAs (U1, U2, U4, U5, and U6). The Drosophila melanogaster SNAP complex (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50, and DmSNAP43) that recognize a proximal sequence element A (PSEA) located approximately 40-60 base pairs upstream of the transcription start site. DmSNAPc recruits RNA polymerase II to the U1-U5 gene promoters, but RNA polymerase III to U6 gene promoters. Interestingly, the precise nucleotide sequence of the PSEA plays a dominant role in determining RNA polymerase specificity of Drosophila snRNA genes. Indeed, there are a few key nucleotide positions that are "conserved-to-be-different" between the PSEAs of fly snRNA genes transcribed by RNA polymerase II and those transcribed by RNA polymerase III. Furthermore, site- specific protein-DNA photo-cross-linking assays have indicated that DmSNAPc adopts different conformations on U1 and U6 promoters. Thus, the investigation of the structural arrangement of DmSNAPc on U1 or U6 PSEAs is important for understanding RNA polymerase specificity. Chapter 1 describes the mapping of protein domains of each of the three DmSNAPc subunits that cross-link to specific phosphate positions in the U1 or U6 PSEAs. To do this, novel methodology was developed that combined the site- specific protein-DNA photo-cross-linking technique with subsequent site-specific chemical cleavage of the protein. The results with DmSNAP190 oriented the subunit with its N -terminus facing the transcription start site of the U1 gene and its C-terminus facing the upstream direction. Chapter 2 describes localization of each of the 4.5 Myb repeats of DmSNAP190 on the U1 PSEA by determining where each cross-links to the DNA. This determined the structural arrangement of the DmSNAP190 Myb repeats on the U1 PSEA. Chapter 3 reports work that determines the structural arrangement of DmSNAP190 Myb repeat domains on the U6 PSEA. The cross-linking data suggest that there is significant movement of at least two of the Myb repeats on U6 promoter sequences relative to U1. These data allow us for the first time to put forth a comprehensive model of the different conformations adopted by DmSNAPc on U1 and U6 promoters