The Myb domain of the largest subunit of SNAPc adopts different architectural configurations on U1 and U6 snRNA gene promoter sequences

Abstract

The small nuclear RNA (snRNA) activating protein complex (SNAPc) is essential for transcription of genes that encode the snRNAs. Drosophila melanogaster SNAPc (DmSNAPc) consists of three subunits (DmSNAP190, DmSNAP50 and DmSNAP43) that form a stable complex that recognizes an snRNA gene promoter element called the PSEA. Although all three subunits are required for sequence-specific DNA binding activity, only DmSNAP190 possesses a canonical DNA binding domain consisting of 4.5 tandem Myb repeats homologous to the Myb repeats in the DNA binding domain of the Myb oncoprotein. In this study, we use site-specific protein–DNA photo-cross-linking technology followed by site-specific protein cleavage to map domains of DmSNAP190 that interact with specific phosphate positions in the U6 PSEA. The results indicate that at least two DmSNAP190 Myb repeats contact the DNA in a significantly different manner when DmSNAPc binds to a U6 PSEA versus a U1 PSEA, even though the two PSEA sequences differ at only 5 of 21 nucleotide positions. The results are consistent with a model in which the specific DNA sequences of the U1 and U6 PSEAs differentially alter the conformation of DmSNAPc, leading to the subsequent recruitment of different RNA polymerases to the U1 and U6 gene promoters.

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

“…DmSNAP50, on the other hand, likely lies more distant from Bdp1 than DmSNAP190 and DmSNAP43. These results are in very good accord with the previously modeled locations of the DmSNAPc subunits on the U6 promoter that were based primarily upon protein-DNA photo-crosslinking (14,29,31,33).…”

“…A model of DmSNAPc, Bdp1, and TBP on a D. melanogaster U6 snRNA gene promoter. The data presented here (from EMSAs, site-specific protein-DNA photocross-linking, and CXMS) allow us to develop a more complete and detailed model that builds upon a series of previous models of DmSNAPc bound to the U6 promoter (14,29,31,33). Together, the data permit us to better integrate DmSNAPc with Bdp1 and TBP.…”

“…Furthermore, we identified an 87-amino-acid region of Bdp1 that was required for Bdp1 to be recruited to the U6 snRNA gene promoter by DmSNAPc (32). Over the years, this has allowed us to build a more and more encompassing picture of the architecture of the protein-DNA complex assembled on the U6 promoter (29,(31)(32)(33).…”

“…Those studies revealed both the linear positions (translational location along the DNA helix) and rotational positions (face of the DNA double helix) occupied by each of the DmSNAPc and TFIIIB subunits on the DNA. Furthermore, by cleaving the DmSNAPc proteins at specific sites after photocross-linking, we were able to identify domains or regions of DmSNAP190, DmSNAP50, and DmSNAP43 that cross-linked to specific nucleotides within or adjacent to the PSEA (29)(30)(31).…”

TFIIIB subunit locations on U6 gene promoter DNA mapped by site‐specific protein–DNA photo‐cross‐linking

“…Double‐stranded U6 DNA probes that each contained a photo‐cross‐linker (azidophenacyl group) located at a specific individual phosphate backbone position either within, upstream, or downstream of the U6 TATA sequence were prepared exactly as described previously in detail . Briefly, two shorter synthetic oligonucleotides were annealed to a longer synthetic oligonucleotide and ligated to form a fully complementary double‐stranded 32 P‐labeled photo‐cross‐linking probe.…”