In vitro trans-activation by chemically synthesized adenovirus region 3 peptides. Reaction properties and mutational analysis

Abstract

The adenovirus E1A gene encodes a protein that transcriptionally activates viral early genes. We have reported that a 49-amino acid chemically synthesized adenovirus type 2 E1A region 3 peptide, PD3 (residues 140-188 of the 289-amino acid protein), can stimulate transcription in vitro from the adenovirus major late promoter. Here we describe reaction properties of E1A trans-activation in vitro with the major late promoter and the early gene 3 promoter and the structural requirements for activity. Stimulation of transcription by PD3 is highest with low levels of DNA template and nuclear extract, and the presence of PD3 eliminates the need to preincubate template with nuclear extract to achieve optimal transcription. These findings suggest that PD3 facilitates a rate-limiting step in the formation of a promoter complex. Analysis of deletion and cysteine substitution mutant PD3 peptides indicates that the C-terminal 70% of the peptide is sufficient for trans-activation in vitro and supports the hypothesis that PD3 contains two functional subregions. The function of one region (residues 140 to about 152) can be overridden under conditions used for in vitro transcription. The second region (residues 153-188) is essential and may function both as a promoter-binding region and as an activating region in vitro.