DNA sequence required for initiation of transcription in vitro from the major late promoter of adenovirus 2.

We have identified a region of the viral genome required for the initiation of transcription in vitro from the major late promoter of adenovirus 2. A fragment of the adenovirus genome containing the cap site of the major late transcripts was inserted into plasmid pBR322 and cloned. Deletions were then generated in vitro in and around the T-A-T-A-A-A-A sequence located 25-31 nucleotides (positions -25 to -31) upstream from the cap site. DNAs with these deletions were tested for their ability to initiate transcription in vitro by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Whereas removal of sequences upstream from position -47 or downstream from position -12 did not abolish transcription, deletions extending into, or beyond, the T-A-T-A-A-A-A sequence reduced transcription to less than 1/10th. Removal of the normal cap site slightly reduced, but did not abolish, transcription. These results indicate that the region of the genome upstream of the cap site, with boundaries within 15-17 nucleotides to either side of the T-A-T-A-A-A-A sequence, is required for the initiation of transcription in vitro from the major late promoter of adenovirus 2.