A protein -DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunlt of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptlde, which corresponds In size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between + 35 and + 47. Another polypeptlde of greater than 205 kd (also a potential TAF) crossllnked preferentially to the region between +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 promoter in nonlnduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent Interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.
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