The transcriptional activity of many sequence-specific DNA binding proteins is directly regulated by posttranslational covalent modification. Although this form of regulation was first described nearly two decades ago, it remains poorly understood at a mechanistic level. The prototype for a transcription factor controlled by posttranslational modification is E. coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Ada repairs methyl phosphotriester lesions in DNA by transferring the aberrant methyl group to one of its own cysteine residues; this site-specific methylation enhances tremendously the DNA binding activity of the protein, thereby enabling it to activate a methylation-resistance regulon. Here, we report solution and X-ray structures of the Cys-methylated chemosensor domain of Ada bound to DNA. The structures reveal that both phosphotriester repair and methylation-dependent transcriptional activation function through a zinc- and methylation-dependent electrostatic switch.

The cI protein of bacteriophage lambda (lambdacI) activates transcription by binding a DNA operator just upstream of the promoter and interacting with the RNA polymerase sigma subunit domain 4 (sigma(4)). We determined the crystal structure of the lambdacI/sigma(4)/DNA ternary complex at 2.3 A resolution. There are no conformational changes in either protein, which interact through an extremely small interface involving at most 6 amino acid residues. The interactions of the two proteins stabilize the binding of each protein to the DNA. The results provide insight into how activators can operate through a simple cooperative binding mechanism but affect different steps of the transcription initiation process.

The nuclear factor of the activated T cell (NFAT) family of transcription factors regulates cytokine gene expression by binding to the promoter/enhancer regions of antigen-responsive genes, usually in cooperation with heterologous DNA-binding partners. Here we report the solution structure of the binary complex formed between the core DNA-binding domain of human NFATC1 and the ARRE2 DNA site from the interleukin-2 promoter. The structure reveals that DNA binding induces the folding of key structural elements that are required for both sequence-specific recognition and the establishment of cooperative protein-protein contacts. The orientation of the NFAT DNA-binding domain observed in the binary NFATC1-DBD*/ DNA complex is distinct from that seen in the ternary NFATC2/AP-1/DNA complex, suggesting that the domain reorients upon formation of a cooperative transcriptional complex.

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The heterologous transcription factors NFAT and AP-1 coordinately regulate cytokine gene expression through cooperative binding to precisely juxtaposed DNA recognition elements. The molecular origins of cooperativity in the binding of NFAT and AP-1 to DNA are poorly understood. Herein we have used yeast one-hybrid screening and alanine-scanning mutagenesis to identify residues in AP-1 that affect cooperative interactions with NFAT on DNA. Mutation of a single conserved Arg residue to Ala in the cJun spacer region (R285A) led to a virtually complete abolition of cooperative interactions with NFAT. The DNA-binding activity of AP-1 alone was unaffected by the cJun R285A mutation, thus indicating that this residue inf luences cooperative binding only. Ala-scanning mutations elsewhere in AP-1, including the cFos subunit, revealed no other strongly interacting single positions. We thus conclude that NFAT contacts AP-1 in the spacer region of the cJun subunit, making an especially important contact to R285, and that these interactions drive formation of the cooperative NFAT͞AP-1͞DNA complex. These results provide a general strategy for selectively ablating cooperativity between transcription factors without affecting their ability to act alone and yield insights into the structural basis for coordinate regulation of gene expression.Antigen recognition by the T-cell receptor results in transmission of a signal across the plasma membrane and activation of mitogenic signaling pathways, leading to the induced expression of interleukin 2 (IL-2) (1). The sequence elements that control antigen-dependent induction of the IL-2 gene, located in an enhancer region within 300 bp upstream of the transcription start point (2), have been extensively studied (1). A sequence in the IL-2 enhancer termed the upstream antigen receptor response element, or ARRE2, serves as a composite recognition site for the nuclear factor of activated T cells (NFAT) and AP-1 (1, 3-6). Whereas AP-1 transcription is induced upon activation of the Ras͞Map kinase pathway (1), NFAT is sequestered in the cytoplasm of resting T cells and translocates to the nucleus in response to Ca 2ϩ mobilization (7). Translocation of NFAT is blocked by the clinically important immunosuppressive drugs cyclosporin A (CsA) and FK506 (7), which inhibit the Ca 2ϩ -activated Ser͞Thr phosphatase calcineurin (8). In the course of T-cell activation, calcineurin is believed to dephosphorylate NFAT directly (9), thereby releasing by an unknown mechanism the blockade to nuclear translocation.Originally isolated from cytoplasmic extracts of a T-cell hybridoma (9), NFAT is now known to define a family of transcription factors containing at least four members (10-14). NFAT family members differ in their tissue distribution-none are localized exclusively in T cells-and appear not to be functionally redundant, as judged by the distinctive phenotype of NFAT1-knockout mice (15, 16). These proteins share a Ϸ300-amino acid domain that may be distantly related to the Rel homology region (...