Simian virus 40 early promoter mutations that affect promoter function and autoregulation by large T antigen

Abstract

A set of nine mutants containing point mutations, and small deletions or insertions, were constructed in the early promoter region of simian virus 40 (SV40) to determine the role of the DNA sequences between the TATA box and the six upstream G + C-rich clusters in early transcription. The mutant templates were tested for transcription activity in vitro in HeLa cell extracts and in vivo in CV-1 and COS cells using the chloramphenicol acetyl transferase gene (CAT) assay. Both in vitro and in vivo results show that the narrow region from nucleotide positions 38 to 41 is an important domain of the early promoter. Deletion and insertion mutations most strongly affect the level of transcription. Specifically a four base-pair deletion in the promoter region enhances the level of transcription four- to sixfold in vitro, but causes a fourfold suppression of CAT gene expression in the in vivo assay. These opposite effects may result from changes in spacing under in vitro and in vivo conditions between the TATA box and the G + C-rich motifs where transcription factors may make simultaneous contact. Of the three T antigen binding sites (I, II and III), sites I and II have already been shown to be involved in the autoregulation of early transcription. Our mutational analyses demonstrate the role of site III, which partially overlaps with nucleotide positions 38 to 41, in the autoregulation of the SV40 early promoter.