A single-base deletion within the protein-coding region of the adenovirus type 5 early region lA (EIA) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an ElA protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress EIA mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of ElA which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated EIA protein does not produce the cis-acting defect in EIA transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 ElA. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for ElA transcription during the late phase of infection.In Escherichia coli, promoter mutations generally result from changes in bases which interact directly with RNA polymerase and occur within 40 bases of the transcription initiation site (62, 68). In contrast to E. coli promoters, the DNA sequences which regulate the transcription of eucaryotic protein-coding genes, which are transcribed by RNA polymerase II, are more complex. The most highly conserved sequence element of polymerase II promoters, the TATA box (11; M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978), centered about 27 bases upstream from the transcription start site, probably interacts directly with the polymerase. Deletion of this sequence usually leads to a reduction in the rate of transcription initiation by a factor of 5 to 10, and the residual transcripts usually have heterogeneous cap sites (18,25,32,34,49,56), which are sites of transcription initiation (15,36). Transcription initiation in vitro in most instances is completely dependent on the TATA sequence (16,40,59,72,78). However, in eucaryotic cells, mutations at much greater distances from the start site than are the TATA box or the -35 region of E. coli promoters can have profound effects on transcription initiation (5, 18, 21, 33-35, 49, 50, 53, 71). Several strong viral promoter regions contain sequences called enhancers, which can increase transcription from a variety of promoters by an unknown mechanism which is largely independent of their position relative to the transcription start site (4,17,21,22,44,45,52,75). Since the function of these more distant transcription control signals is largely independent of their distance from the transcription initiation site, it seems unlikely that they interact directly with RNA polymerase II (44,48,52).In most genes transcribed by RNA polymerase II which have been analyzed, DNA sequences required for transcription initiation lie upstream from th...