Mapping the Interactions of the Multi-Subunit General Transcription Factor IIIB (TFIIIB) with U6 Promoter DNA by Site-Specific Protein-DNA Photo-Cross-Linking
- Author(s): KANG, JIN JOO
- Advisor(s): Stumph, William E
- et al.
U6 small nuclear RNA (snRNA) functions as a component of the spliceosome and is required to splice eukaryotic pre-mRNAs. Although the other spliceosomal snRNAs are synthesized by RNA polymerase II, U6 snRNA is synthesized by RNA polymerase III. The genes that code for U6 snRNA have promoters that are completely gene-external, that is, in the 5'-flanking DNA. These promoters contain a TATA box, as well as a required upstream proximal sequence element (PSE) that is unique to snRNA genes. The PSE is recognized by a multi-subunit transcription factor SNAPc, whereas the TATA sequence is bound by the general transcription factor TFIIIB, which is responsible for assembling an RNA polymerase III initiation complex. In the fruit fly Drosophila melanogaster, TFIIIB is composed of three distinct subunits that assemble on the U6 promoter: TBP, Bdp1 and Brf1. [On tRNA and 5S rRNA promoters in fruit flies, TRF1 (TBP-related factor 1) is employed in place of TBP.]
At the present time, the overall structures of TFIIIB and particularly the Brf1 and Bdp1 subunits are not well known. To better understand the mode of fruit fly TFIIIB binding to DNA, I have used site-specific protein-DNA photo-cross-linking. I introduced photo-cross-linker at every second phosphate position in the DNA backbone on both the template and non-template strands within and surrounding the fly U6 TATA box. I have in this manner mapped the locations where each of the TFIIIB subunits lies in close proximity to the DNA. Brf1 cross-links strongly to the DNA within and upstream of the TATA box, whereas Bdp1 cross-links strongly both upstream and downstream but not within the TATA sequence. The data indicate that Bdp1 lies on the DNA in close proximity to two subunits of DmSNAPc. Bdp1 may therefore play a primary role in the recruitment of TFIIIB to the U6 promoter through interactions with DmSNAPc.