Description
The goal of this study involves determining the requirements and mechanisms of RNA polymerase-specific transcription complex assembly on U6 small nuclear RNA (snRNA) gene promoters in Drosophila melanogaster. In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB and a proximal sequence element, PSE, recognized by the small nuclear RNA activating protein complex (SNAPc). In Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. Tjian’s lab [Takada et al., 2000] concluded that TRF1 (TBP-related factor 1), instead of TBP, was utilized at all Pol III promoters (including U6 transcription) in D. melanogaster . However, preliminary work in our lab on U6 caused us to question this widely accepted notion. Chapter 1 elucidates the transcriptional requirements of Drosophila melanogaster U6 snRNA genes with reference to solve the discrepancy between the two general transcription factors (GTFs), TBP and TRF1. Our results revealed that although TRF1 mediates Pol III-dependent transcription of the tRNA genes, transcription of the U6 snRNA genes in contrast utilizes TBP [Verma et al., 2013] indicating the differential utilization of TBP and TRF1 at different classes of Pol III promoters. Chapter 2 describes a study that provided a mechanistic explanation of the Pol III specificity of U6 snRNA gene promoters by investigating the existence of interactions between DmSNAPc and TFIIIB on the U6 promoter. Our results showed that DmSNAPc, when bound to a U6 promoter (but not to a U1 promoter), attains the ability to recruit Bdp1 and subsequently TBP for Pol III transcription. Chapter 3 studies various truncation and alanine-scanning mutations of DmSNAPc in an attempt to identify regions involved in the novel interaction between DmSNAPc and Bdp1 on the U6 promoter as reported in Chapter 2. Despite utilizing a variety of alanine-scanning constructs of DmSNAP43 (the subunit of DmSNAPc that cross-links to the DNA furthest downstream of the U6 PSEA and hence closest to TFIIIB binding site on the U6 promoter), we were unable to detect a region responsible for Bdp1 recruitment.
