Genetic Analysis of the Bovine Papillomavirus E2 Transcriptional Activation Domain

Abstract The bovine papillomavirus type 1 E2 transactivator has a large amino-terminal 215-residue transcriptional activation domain (TAD) that is active in Saccharomyces cerevisiae and higher eukaryotic cells. Comparison to other transcriptional activators suggests that its functions may be mediated in part through two acidic regions, A1 and A2, in this domain. We have characterized the functional elements within the E2 TAD using LexA–E2 fusions and by screening randomly generated libraries of E2 mutations for transcriptional activation in yeast. The A1 region was highly sensitive to substitutions that reduce negative charge, although there was not a perfect correlation between overall charge and transcriptional activity. Mutations were isolated within a hydrophobic amino acid motif that overlaps the A2 region and resembles elements described in other viral and cellular transactivation domains. When fused to the LexA DNA binding domain, this hydrophobic motif within the acidic A2 region was unable to activate transcription in S. cerevisiae. Multiple highly defective mutations primarily altering hydrophobic amino acids were identified in the distal third of the E2 TAD. The transcription phenotype of many of these E2 TAD mutations was similar in yeast and COS cells.