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J. Biol. Chem., Vol. 263, Issue 21, 10377-10385, 07, 1988

Identification of a downstream sequence and binding protein that regulate adenovirus major late promoter transcription in vitro

RB Cohen, L Yang, JA Thompson and B Safer
Section on RNA and Protein Biosynthesis, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.

Gel electrophoresis mobility shift and DNase I footprint assays detect a cellular nuclear protein in extracts made from uninfected human cells which binds to a downstream promoter sequence (DPS) in the human adenovirus 2 major late promoter. By DNase I footprint and mutation analyses, we have determined that this new regulatory element extends from positions +146 to +165 (relative to the cap site at position +1). We show by UV cross-linking that a 40-kDa polypeptide specifically binds to this region. Mutations within the DPS which decrease protein binding by 80-90% also cause a 2.5-3-fold decrease in in vitro major late promoter transcription efficiency. Alteration of the template in the 5'-flanking region of the DPS does not affect nuclear protein binding or transcription efficiency. Interestingly, a T----G transversion at position +160 which increases protein binding also impairs promoter activity.
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