Generation and functional analyses for base-substitution mutants of the adenovirus 2 major late promoter

The function of guanosine residues surrounding the TATA box of the adenovirus 2 major late promoter (MLP) in promoting efficient transcription initiation and in selecting a specific transcription start site in vitro has been examined. Multiple and single base substitutions (G----A) were generated in this region (from -63 to +25 relative to the cap site, +1) of the MLP. The promoter activities of the wild type and 21 mutants were assayed in an in vitro transcription system using whole cell extract (WCE) prepared from HeLa cells. The results suggest that the strings of G residues immediately adjacent to the TATA box are not required for full promoter activity in vitro. These G residues also appear not to be involved in the selection of a specific transcription start site by RNA polymerase II in vitro, since the identical cap site was used by wild-type and mutated MLP's. However, two G residues (-55 and -57) were identified as part of an upstream promoter element: a G----A transition at either -55 or -57 resulted in a 2.6 fold reduction in promoter activity. However, neither single nor double G----A transitions at -62 and -63 had an effect on promoter activity.