Structural and functional studies of the Drosophila melanogaster small nuclear RNA activating protein complex (DmSNAPc)

Abstract

The small nuclear RNA activating protein complex (SNAPc) is an evolutionarily conserved multi‐subunit factor required for transcription of the spliceosomal small nuclear RNA (snRNA) genes by both RNA polymerase II (U1, U2, U4, and U5) and RNA polymerase III (U6). Three distinct polypeptides have been identified as subunits of D. melanogaster SNAPc; however, the stoichiometry of these three subunits in DmSNAPc had not been investigated. By co‐expressing each subunit with two different tags and by doing band‐shift and super‐shift analyses, we have determined that DmSNAPc is a heterotrimer with a 1:1:1 subunit stoichiometry. DmSNAPc recognizes an ~21 bp long DNA sequence denoted the PSEA about 40‐60 bp upstream of the transcription start site. Interestingly, the PSEAs of the U1 and U6 genes are not interchangeable even though they are identical at 16 of 21 nucleotide positions. In fact, changing the U1 PSEA to a U6 PSEA inactivated the U1 promoter. We have now found that this substitution does not affect the association of DmSNAPc with the promoter; instead, it disrupts the recruitment of TBP. This finding is consistent with a working model in which DmSNAPc binds in different conformations to the U1 and U6 PSEAs. We believe these conformational differences in DmSNAPc lead to differential RNA polymerase selectivity at the U1 and U6 promoters. Supported by NSF and in part by the California Metabolic Research Foundation.