In order to gain insight into requirements for template activation and commitment in mammalian transcription, TATA site occupancy was measured in native SV40 viral transcription complexes that were in the process of transcription elongation at the time of cell lysis. This was accomplished by quantifying resistance to restriction enzyme digestion of transcription complexes in nuclear lysate. The rate of cleavage at the TATA site of the late gene in the native complex was slower than that of a bare DNA control, both for wild-type virus and for a virus containing a TATA consensus sequence. These results suggest that the TATA site in the transcription elongation complex in vivo is occupied with transcription factor TBP/TFIID. When considered in light of previous work, these findings support a model in which transcription activation involves reinitiation from a promoter that contains both activator and TFIID bound in a stable complex.
Copyright 2001 Academic Press.