The zinc finger region of the adenovirus E1A transactivating domain complexes with the TATA box binding protein.

Abstract

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...

C-terminal-binding protein interacting protein binds directly to adenovirus early region 1A through its N-terminal region and conserved region 3

“…It is notable that mutation of the two N-terminal cysteines of the Zn 2 þ -finger (for example, in dl 139-160) do not negate CtIP interaction -therefore it seems likely that binding of the metal ion per se is not required for the interaction. TBP binds to CR3 through amino acids 147, 150, 157 and 171-174 (Geisberg et al, 1994) -the latter three residues falling in the site necessary for CtIP interaction. Similarly, mutation of several amino acids within CR3, which do not interfere with TBP binding, blocks transcriptional activation.…”

“…CtIP bound to all CR3 polypeptides, except for 169-177 from which the C-terminal half of the Zn 2 þ -finger is deleted (Supplementary Figure 2b). This region is also required for AdE1A's binding to TBP and Sur2 (Geisberg et al, 1994;Boyer et al, 1999;Wang and Berk, 2002). However, it is interesting to note that the general level of binding to the mutant CR3s was appreciably reduced compared to wt except for CR3YF175 (Supplementary Figure 2b).…”

“…Binding of the Rb family or CBP/p300 is necessary for AdE1A to promote S phase entry, but binding of both sets of proteins is required for AdE1A-mediated transformation (Egan et al, 1988;Jelmsa et al, 1989;Howe et al, 1990;Wang et al, 1993). CR3 contains a zinc (Zn 2 þ )-finger motif and is the site of interaction with a number of proteins involved in transcriptional regulation (Culp et al, 1988;Geisberg et al, 1994Geisberg et al, , 1995Rasti et al, 2006). These interactions with proteins such as TATA-binding protein (TBP), associated transcription factor (ATFs), suppressor of ras (Sur2) mediator 23 (MED23), proteasomal components and trans-acting factors are necessary for transcriptional activation and for the expression of other viral early region proteins (reviewed by Jones, 1995;Avvakumov et al, 2004).…”

Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway

“…For example, the Rb family of transcriptional corepressors bind to CR1 and CR2, the CBP/p300 family of acetyltransferases bind to the N-terminal region and CR1, whereas CtBP1 and CtBP2 associate with the C-terminal CR4 domain (Whyte et al, 1989;Dyson et al, 1992;Boyd et al, 1993;Eckner et al, 1994;Arany et al, 1995). CR3, exclusive to the E1A13S protein, binds to proteins involved in transcriptional activation, such as TATA binding protein (TBP), Mediator 23 (Med23) and associated transcription factors (ATFs), various transcription factors and proteasome components (Lee et al, 1991;Green, 1990, 1994;Geisberg et al, 1994Geisberg et al, , 1995Boyer et al, 1999;Wang and Berk, 2002;Rasti et al, 2006).…”

Adenovirus early region 1A protein binds to mammalian SUG1-a regulatory component of the proteasome

“…The binding sites on E1A for these and other cellular proteins have been mapped in most cases to regions of E1A conserved between di erent virus serotypes (Moran and Mathews, 1987). Thus, for example, E1A interacts with the pRb family through conserved regions 1 and 2 (CR1 and 2) (Whyte et al, 1989), with TBP binding to CR3 (Lee et al, 1991;Horikoshi et al, 1991;Geisberg et al, 1994) and the C terminal binding protein binding to a short highly conserved region very close to the C terminus (Boyd et al, 1993).…”