TRA1P IS A COMPONENT OF THE YEAST ADA-CENTER-DOT-SPT TRANSCRIPTIONAL REGULATORY COMPLEXES

The yeast Ada and TBP class of Spt proteins interact in multiple compl exes that are required for transcriptional regulation. We have identif ied Tra1p as a component of these complexes through tandem mass spectr ometry analysis of proteins that associate with Ngg1p/Ada3p, TRA1 is a n essential gene and encodes a 3744-amino acid protein that is a membe r of a group of proteins including the catalytic subunit of DNA-depend ent protein kinase, ATM and TRRAP, with carboxyl-terminal regions rela ted to phosphatidylinositol 3-kinases, The interaction between Tra1p a nd Ada/Spt components was verified by the reciprocal coimmunoprecipita tion of Ada2p and Tra1p from whole cell extracts in one or more comple xes containing Spt7p, Tra1p cofractionated with Ngg1p and Spt7p throug h consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. Th e association of Tra1p with two Ada Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q colum n. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within i mmunoprecipitates of Tra1p or the Ada.Spt complexes.