Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus ooeytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.
All Science Journal Classification (ASJC) codes
- Molecular Biology