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Description
Small nuclear RNAs (snRNAs) are active constituents in eukaryotic cells and perform functions such as RNA processing and transcription regulation. Some of these snRNAs (e.g. U1, U2, U3, U4 and U5) are synthesized by RNA polymerase II (Pol II), whereas others (e.g. U6) are synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster the transcription process for both U1 and U6 (as examples) is dependent upon a unique 21 base pair DNA sequence called the proximal sequence element A, or PSEA, which resides approximately 40 to 60 base pairs upstream from the snRNA transcription start site. The PSEA is well-conserved in promoters of the Pol II transcribed genes (U1, U2, U4 & U5), however U6 PSEAs contain several nucleotides that are different from the Pol II transcribed gene set. During transcription the PSEA of both U1 and U6 is recognized by a heterotrimeric transcription factor called the Drosophila melanogaster snRNA activating protein complex (DmSNAPc) that consists of three polypeptide subunits called DmSNAP43, DmSNAP50 and DmSNAP190. Prior studies have shown that DmSNAPc takes on distinctive conformations when bound to either the U1 or U6 PSEA, which subsequently leads to the recruitment of either Pol II or Pol III. A critical component of DmSNAPc's ability to recognize and bind to the U1 and U6 PSEA is a cluster of Myb repeats within the DmSNAP190 protein. Myb repeats are known DNA binding motifs that were first characterized in the Myb oncoprotein. The objective of the research presented here is to localize where each of the Myb repeats in DmSNAP190 cross-links to the U1 PSEA. This was done by performing site-specfic protein-DNA photo-crosslinking experiments followed by hydroxylamine cleavage of the DmSNAP190 protein. The products were then identified by electrophoresis and autoradiography. From these experiments I was able to determine that DmSNAP190 binds to the snRNA promoter region with its N-terminus directed towards the transcription start site while its C-terminus is located towards the 5' end of the PSEA. Furthermore, my results determined where each of the 4.5 Myb repeats of DmSNAP190 contact the DNA on the U1 PSEA. The two C-terminal repeats (Rc and Rd) appear to contact the most 5' region of the PSEA complex in a manner consistent with those observed in other Myb-DNA complexes. Interestingly, the Ra and Rb repeats appear to contact a more central region of the PSEA in a near canonical manner similar to Rc and Rd. Rh, on the other hand, appears to contact the 3' end of the PSEA in a non-canonical fashion.
