Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Structural and Functional Studies of the Drosophila Melanogaster Small Nuclear RNA Activating Protein Complex (DmSNAPc)
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 in vivo. 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 model in which DmSNAPc binds in different conformations to the U1 and U6 PSEAs and that these conformational differences in DmSNAPc lead to differential RNA polymerase selectivity at the U1 and U6 promoters. All three subunits of DmSNAPc contact DNA and are required for its sequence-specific DNA binding activity, but only one of the subunits contains a canonical DNA-binding domain. We have recently identified domains in each of the three subunits that are required for assembly of the DmSNAP complex and for its DNA-binding activity. This work was supported by NSF and in part by the California Metabolic Research Foundation.
William E. Stumph
Chemistry and Biochemistry and Molecular Biology Institute