Functional analysis of the nucleotide sequence surrounding the cap site for adenovirus type 5 region E1A messenger RNAs

Abstract

We have constructed a set of small, dispersed deletion mutations in the sequences surrounding the cap site of the adenovirus type 5 region E1A transcription unit. The effects of the mutations on E1A transcription were studied in vitro using a HeLa cell-free extract, and in vivo by reconstructing the mutations back into intact viral chromosomes and analyzing E1A messenger RNAs synthesized after infection of HeLa cells. The sequence between -35 and +20 (relative to the cap site at +1) was important for efficient E1A transcription and cap site selection in vitro. This region includes the "TATA" homology, which appeared essential for transcription. Sequences upstream of -35 were dispensable for transcription in vitro. Different results were found upon analysis of the same set of deletions in vivo. None of the mutations affected the steady-state levels of cytoplasmic, E1A-specific mRNAs found in infected HeLa cells by more than twofold. Deletions of the TATA homology, however, generated E1A mRNAs with heterogeneous 5' ends, and deletions downstream of the homology displaced the 5' end of mRNAs by about the size of the deletion.