In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals.

A vaccinia virus gene that is expressed throughout the reproductive cycle was found to have two sets of RNA start sites approximately 55 nucleotides apart. The site nearest to the coding segment is used early in infection and the one further upstream is used after DNA replication. A series of 5' to 3' deletions were made in the promoter region, and the truncated DNA segments were then ligated to the coding portion of the procaryotic chloramphenicol acetyltransferase gene to measure expression. The effects of these mutations on chloramphenicol acetyltransferase synthesis were determined in a vaccinia virus helper-dependent transient expression system and by forming infectious vaccinia virus recombinants that contain the chimeric genes. Deletions extending up to 31 nucleotides before the late RNA start site had no effect on either early or late expression. Removal of an additional 15 nucleotides produced a dramatic decrease in late expression but had no effect on early expression. The latter was not diminished until the deletion was extended from 31 to 24 nucleotides before the early RNA start site. These results were confirmed by transcriptional analyses. We concluded that this vaccinia virus gene has two promoters and that the regulatory signals for each are located within 31 nucleotides of their sites of transcription.