The 289R EMA protein of adenovirus transactivates a variety of viral and cellular promoters through protein-protein interactions. In earlier studies, mutational analyses of the ElA transactivating domain identified residues that are critical for transactivation and implied that the zinc finger region of the transactivating domain binds a transcription factor. Also, the ElA activation domain was found to bind to the TATA box binding protein (TBP) in vitro. Here, we tested the significance of the E1A-TBP interaction for ElA transactivation by analyzing the effects of conservative substitutions at each of the 49 residues of the ElA activation domain. Seven of the substitutions significantly diminished TBP binding in vitro. All of these were in the zinc finger region and were defective for transactivation in vivo. The perfect correlation between reduced TBP binding and transactivation argues strongly that a direct interaction between the ElA activation domain and TBP is critical to the mechanism of ElA activation. This genetic analysis leads us to further suggest that another factor, which is limiting, is also necessary for ElA-mediated transactivation.The adenovirus ElA 289R protein is a potent transactivator of a variety of viral and cellular promoters (reviewed in refs. 1 and 2). The absence of a common cis element in E1A-inducible promoters (1) and the weak, nonspecific DNA binding activity of the 289R protein (3) suggests that ElA stimulates transcription through protein-protein interactions with cellular transcription factors.The transactivation function of the 289R protein maps to an internal stretch of 49 amino acids (residues 140-188) (4, 5). The salient feature of the ElA transactivating domain is a metal binding structure that is formed by four cysteine residues, which coordinate a single zinc atom (6, 7). The importance of a structurally intact Cys4 zinc finger is highlighted by the fact that replacing any of the four cysteine residues produces a mutant protein that is incapable of activating transcription.Individually substituting every amino acid in the transactivating domain identified additional residues that are also critical for transactivation (8). From this study, ElA proteins containing mutations in a contiguous stretch of residues C terminal to the zinc finger were found not only to be defective for transactivation but also to display a strong transdominant negative phenotype. Furthermore, this study revealed that the transactivating domain is composed of two functionally distinct regions, a finger region (residues 147-177) and a carboxyl region (residues 183-188), each of which is postulated to bind to a different cellular protein. Indeed, two distinct classes of factors, the TATA box binding protein (TBP) and the activating transcription factor family (ATF), have been recently shown to directly interact with the transactivating domain of ElA (9, 10, 34). Taken together, these studies suggest that ElA transactivates by interacting with sequence-specific DNA binding transcription factor...

Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex.