Description
The small nuclear RNAs (snRNAs) are an essential class of non-coding RNAs and play important roles in gene expression and cell growth. All snRNA gene transcription is dependent upon the same multi-subunit transcription factor, called the snRNA Activating Protein complex (SNAPc). In metazoans, the U6 small nuclear RNA (snRNA) gene has promoters that consist of a proximal sequence element A (PSEA) recognized by SNAPc and a TATA box recognized by the TATA-binding protein (TBP). In the fruit fly Drosophila melanogaster (Dm), SNAPc has 3 subunits: DmSNAP190, DmSNAP50, and DmSNAP43. On the U6 promoter, DmSNAPc recruits Pol III general transcription factors, including TBP, Brf1, and Bdp1. DmSNAPc can directly recruit Bdp1 to the U6 promoter. Interestingly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSEA rather than a U1 PSEA. Furthermore, an 87-residue region of Bdp1 (Bdp1(424-510)) is essential for this recruitment and sufficient to recruit TBP to the TATA box. Cross-linking mass spectrometry (CXMS) was utilized to better understand the interactions between DmSNAPc and Bdp1 on the U6 gene promoter. As a result, most of the CXMS crosslinks resided in the "core" region of DmSNAPc that was composed of the interface among DmSNAP50, DmSNAP43, and the N- terminal region of DmSNAP190. Also, the non-conserved N- and C-terminal regions of DmSNAP190 and the non-conserved C-terminal region of DmSNAP43 are located in close proximity to Bdp1, but not DmSNAP50. DmSNAPc and Bdp1 models were generated by RaptorX. Interestingly, the C-terminus of DmSNAP190 happens to be structurally related to the ligand-binding domains of members of the nuclear hormone receptor superfamily. The location of this domain suggests a possibility that the activity of DmSNAPc and the expression of snRNA genes could be regulated by an unknown small organic molecule of intracellular or extracellular origin. The results from CXMS, coupled with the published data from site-specific protein-DNA photo-cross-linking, were used to generate a model of DmSNAPc on the U6 promoter. Although a Bdp1 model was generated by RaptorX, there was insufficient information to place the predicted structure of Bdp1 onto the DmSNAPc-U6 promoter model.
