Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster

ABSTRACT

U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

KEYWORDS:

• SNAPc-Bdp1 interaction
• TBP recruitment
• transcription factor assembly
• U6 preinitiation complex
• U6 small nuclear RNA transcription

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation (MCB-1616487) and by the California Metabolic Research Foundation.

We thank Phil Gafken and Lisa Jones at the Fred Hutchinson Cancer Research Center proteomics facility for mass spectrometry analyses.