Transcriptional regulation by a point mutant of adenovirus-2 E1a product lacking DNA binding activity.

Abstract

The adenovirus E1a protein (E1A) regulates transcription through interaction with transcription factors bound to DNA, like cAMP response element BP1/ATF2, or through dissociating E2F transcription factor complex. However, it was also reported that E1A can bind to DNA (Chatterjee, P. K., Bruner, M., Flint, S. J., and Harter, M. L. (1988) EMBO J. 7, 835-841), and it is not clear whether DNA binding of E1A is involved in a part of the process of transcriptional regulation by E1A. In this paper, the small region of E1A that is responsible for DNA binding was identified and a point mutant lacking DNA binding activity was constructed. Analysis of deletion mutants of E1A proteins expressed in bacteria showed that a basic region between amino acids 201 and 216 of E1A is essential for DNA binding. Point mutation of arginines at amino acid numbers 205 and 206 to aspartic acids completely abolished the DNA binding activity of E1A. Using this mutant, the requirement of the E1A DNA binding for E1A-dependent transcriptional regulation was examined. trans-Activation of the adenovirus E4 promoter and trans-repression of the human c-erbB-2 promoter by this point mutant were examined by cotransfection experiments. Mutations of the E1A DNA-binding domain affected neither the E1A-induced trans-activation nor trans-repression at all. These results give complete proof that the DNA binding activity of E1A is not required for transcriptional regulation by E1A.