A multicomponent cis-activator of transcription of the E1b gene of adenovirus type 5

We report that transcription of the adenovirus type 5 E1b gene is activated substantially in cis by sequences located between positions -362 and -49 with respect to the RNA start site. DNA fragments consisting of the -362 to -49 sequences, or subsets thereof, were inserted into a reporter plasmid containing a minimal E1b promoter (positions -48 to +14) joined to the Escherichia coli cat gene. In the presence of cotransfected E1a and E1b genes in trans, CAT enzyme synthesis in transfected KB cells was stimulated about 20-fold by sequences from -362 to -49 (XY) in cis and to a lesser extent by sequences from either -362 to -128 (X) or -127 to -49 (Y). Adenoviruses were isolated lacking the X, Y, or XY sequences and KB cells were infected with one of the mutants, as well as wild-type virus to provide E1a and E1b in trans. Deletion of both X and Y resulted in a 20-fold reduction in early E1b RNA and a 12-fold reduction in late RNA. Deletion of X or Y alone produced up to 5-fold reductions in early or late E1b RNA accumulation. In vitro DNA-protein interactions in the Y sequence were revealed by modification of the procedure used for previous detection of X region footprints. These data indicate that X and Y sequences, which include E1a protein coding and 3' untranslated DNA, also participate in DNA-protein interactions necessary for high levels of E1b promoter activity. The presence of such overlapping genetic elements raises the interesting possibility that functional E1a and E1b mRNAs must be synthesized from separate templates.