PROTEIN-DNA CROSS-LINKING OF A TFIID COMPLEX REVEALS NOVEL INTERACTIONS DOWNSTREAM OF THE TRANSCRIPTION START

A protein - DNA complex containing TFIID has been analyzed by crosslin king. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated fa ctors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 k d (also a potential TAF) crosslinked preferentially to the region betw een +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 p romoter in noninduced cells. Crosslinking revealed that as the salt co ncentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element we re disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinit y of the start site and the region immediately downstream. A protein c ontact downstream of +35 might function in pausing polymerase.