UC San Diego

In eukaryotes, small nuclear RNAs (snRNAs) are required for pre-mRNA splicing. Most snRNAs, such as U1, U2 ,U4 and U5, are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. Transcription of snRNA genes by either RNA polymerase is dependent upon a proximal sequence element (PSE) centered approximately 50- 55 base pairs upstream of the start site. The PSE is recognized by the small nuclear RNA activating protein complex (SNAPc), a multi-subunit transcription factor. In Drosphila melanogaster, the PSE is more specifically termed the PSEA to distinguish it from a second conserved element termed the PSEB present in the promoter of the Pol II transcribed fly snRNA genes. Interestingly, the fly U1 and U6 PSEAs are not functionally interchangeable, even though both are recognized by the same protein, DmSNAPc. A five-nucleotide substitution that changed the U1 PSEA to a U6 PSEA was shown to inactivate the U1 promoter. In light of this knowledge I wished to investigate why the U6 PSEA cannot functionally substitute for the U1 PSEA. I sought to determine whether the U6 PSEA substitution disrupts a specific step in RNA polymerase II transcription pre- initiation complex assembly in vivo. To accomplish this, I used a chromatin immunoprecipitation (ChIP) assay. In chapter 1, I describe the preparation of reagents needed for the ChIP assays. I expressed TBP and two of the three subunits of DmSNAPc in bacteria. I then purified the proteins and used them for polyclonal antibody production. In chapter 2, I demonstrate that the antibodies can be used in ChIPs to detect DmSNAP43, DmSNAP50 and TBP bound to the endogenous U1 promoter in vivo. I then generated cell lines stably transfected with reporter constructs for the U1 wild type promoter or the U1 promoter that contained a U6 PSEA. Interestingly, my ChIPs indicated that DmSNAPc assembled on both types of promoters. On the other hand, TBP assembled only on the wild type promoter. These results are consistent with a model in which DmSNAPc assumes a conformation on the U6 PSEA that prevents the assembly of a Pol II transcription pre-initiation complex