The 289R ElA protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by ElA is mediated through cellular transcription factors. In particular, the ability of the trans-dominant ElA point mutant hr5 (Ser-185 to Asn) to inhibit wild-type ElA trans-activator (40, 49, 63). The independent nature of the trans-activating domain was revealed by the ability of a synthetic peptide comprising the 49-amino-acid domain to stimulate transcription from an early viral promoter in microinjection experiments (31).The salient structural feature of the trans-activating domain is a C4-type zinc finger sequence defined by C154, C157, C171, and C174 (7). We have shown that the 289R protein binds a single zinc ion. Since ElA does not bind DNA directly, it is different from other regulatory proteins which mediate DNA binding through their zinc fingers, e.g., TFIIIA (16,39), Spl (21), and the glucocorticoid receptor (11). However, the zinc finger of ElA is clearly an essential component of the trans-activating domain, since individually substituting glycine or serine for each of the four cysteine residues destroyed trans-activation (7). Substituting serine for C157, C171, or C174 also destroyed zinc binding, whereas replacing C154 with serine surprisingly had no effect on zinc binding. X-ray absorption fine structure analysis showed that the zinc in the wild-type (wt) 289R protein is indeed coordinated to four cysteine residues; the single amino acid substitution of serine for C154 resulted in the recruitment of two histidines on the left side of the finger to bind zinc in conjunction with C171 and C174 (59). These results argue that for trans-activation to occur, zinc must be bound by ElA in a specific way.Following our report of the identification of an ElA trans-dominant point mutant, hr5 (185SN) (13), mutant forms of several other viral trans-activator proteins which also exhibit trans-dominance have been identified, including Tax of human T-cell lymphotropic virus type 11 (58), VP16 of herpes simplex virus type 1 (57), and Rev and Tat of human 4287

The ElA gene of adenovirus type 5 encodes two major proteins of 289 and 243 amino acid residues, which are identical except that the larger protein has an internal stretch of 46 amino acids required for efficient trans-activation of early viral promoters. This domain contains a consensus zinc finger motif (Cys-Xaa2-Cys-Xaal3-Cys-Xaa2-Cys) in which the cysteine residues serve as postulated ligands. Atomic absorption spectrophotometry applied to bacterially expressed ElA proteins revealed that the 289-amino acid protein binds one zinc ion, whereas the 243-amino acid protein binds no zinc. Replacing individual cysteine residues of the finger with other amino acids destroyed the trans-activating ability of the 289-amino acid protein, even when structurally or functionally conserved amino acids were substituted. These results strongly suggest that the zinc finger of the 46-amino acid domain is intimately linked to the ability of the large EMA protein to stimulate transcription of ElA-inducible promoters. Furthermore, zinc binding to one of the mutant finger proteins suggests either that only a precise finger structure formed by the tetrahedral coordination of zinc to the four consensus ligands is required for trans-activation or, possibly, that one of several neighboring histidine residues in various combinations with three of the consensus cysteine residues normally coordinates zinc. How the zinc finger in ElA might interact with DNA or protein to bring about trans-activation is discussed.The ElA gene ofadenovirus functions normally by transcriptionally activating (trans-activating) other early viral promoters, which leads to productive infection (1, 2). ElA gene products can also trans-activate the promoters of some cellular genes-e.g., heat shock and f3-tubulin (3, 4)-and, conversely, can repress enhancer-stimulated transcription from the promoters of certain other genes-e.g., immunoglobulin and simian virus 40 early promoters (5-7). How these modes of positive and negative regulation by ElA may be involved in ElA-dependent viral transformation and tumorigenesis (reviewed in ref . 8)

Trans-activation by the adenovirus E1A 289R protein requires a zinc finger defined by Cys-154, Cys-157, Cys-171, and Cys-174. Whereas individually replacing the four cysteine residues with serines resulted in a loss of transactivation, only three of the Cys----Ser mutants (C157S, C171S, and C174S) lost the ability to bind Zn(II). X-ray absorption fine structure analysis revealed that, in the wild-type protein, Zn(II) is coordinated by four cysteine residues whereas in the C154S mutant, Zn(II) is coordinated by two histidines and two cysteines. The mutant protein probably retains, as ligands, two cysteines on the right side of the zinc finger (Cys-171 and Cys-174) and recruits two of the four histidines on the left side (His-149, His-152, His-158, and His-160), despite the presence of Cys-157. This finding may shed light on the general structural requirements of zinc fingers.

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The 289R E1A protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by E1A is mediated through cellular transcription factors. In particular, the ability of the trans-dominant E1A point mutant hr5 (Ser-185 to Asn) to inhibit wild-type E1A trans-activation was proposed to result from the sequestration of a cellular factor. Using site-directed mutagenesis, we individually replaced every residue within and flanking the trans-activating domain with a conservative amino acid, revealing 16 critical residues. Six of the individual substitutions lying in a contiguous stretch C terminal to the zinc finger (carboxyl region183-188) imparted a trans-dominant phenotype. trans-Dominance was even produced by deletion of the entire carboxyl region183-188. Conversely, an intact finger region147-177 was absolutely required for trans-dominance, since second-site substitution of every critical residue in this region abrogated the trans-dominant phenotype of the hr5 protein. These data indicate that the finger region147-177 bind a limiting cellular transcription factor and that the carboxyl region183-188 provides a separate and essential function. In addition, we show that four negatively charged residues within the trans-activating domain do not comprise a distinct acidic activating region. We present a model in which the trans-activating domain of E1A binds to two different cellular protein targets through the finger and carboxyl regions.