DNA sequences required for trans-activation of an immediate-early frog virus 3 gene

Abstract

A plasmid containing 78 bp of the promoter region of the immediate-early frog virus 3 (FV3) gene ICR 169 placed 5′ to the coding sequences for chloremphenicol acetyltransferase (CAT) can only be induced to synthesize CAT after transfection in the presence of FV3. To determine what DNA sequences in the promoter were required for virus-induced transcription, I used site-directed mutagenesis to construct deletions and point mutations throughout the promoter region. The mutant promoters were then analyzed for their ability to be induced by FV3. Deletion of 27 by from the 5′ end of the promoter had little effect on FV3-induced CAT synthesis. Although deletion of, and point mutations within, the 7-bp TATA-like box reduced CAT synthesis to 16–50% of that obtained with the wild-type promoter, only deletion of the 7-bp sequence caused a detectable shift of the transcription start site, indicating that the function of the AT-rich region is to position the RNA polymerase. The most significant reduction in CAT synthesis—to 1.5% of wild-type—occurred after deletion of the 23-bp immediately 5′ to the TATTTTA box, which marks this 23-bp sequence as the critical cis-regulatory element for FV3 trans-activation.