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.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , RNA, Small Nuclear/genetics , TATA-Box Binding Protein/metabolism , Transcription Factor TFIIIB/metabolism , Animals , Drosophila melanogaster/genetics , Promoter Regions, Genetic , Protein Binding , Protein Interaction Maps , Protein Subunits/metabolismABSTRACT
In metazoans, U6 small nuclear RNA (snRNA) gene promoters utilize a proximal sequence element (PSE) recognized by the small nuclear RNA-activating protein complex (SNAPc). SNAPc interacts with the transcription factor TFIIIB, which consists of the subunits TBP, Brf1 (Brf2 in vertebrates), and Bdp1. Here, we show that, in Drosophila melanogaster, DmSNAPc directly recruits Bdp1 to the U6 promoter, and we identify an 87-residue region of Bdp1 involved in this interaction. Importantly, Bdp1 recruitment requires that DmSNAPc be bound to a U6 PSE rather than a U1 PSE. This is consistent with the concept that DmSNAPc adopts different conformations on U6 and U1 PSEs, which lead to the subsequent recruitment of distinct general transcription factors and RNA polymerases for U6 and U1 gene transcription.
