TATA-binding protein-associated factor 1 (TAF1) is an essential component of the general transcription factor IID (TFIID), which nucleates assembly of the preinitiation complex for transcription by RNA polymerase II. TATA-binding protein and TAF1⅐TAF2 heterodimers are the only components of TFIID shown to bind specific DNA sequences (the TATA box and initiator, respectively), raising the question of how TFIID localizes to gene promoters that lack binding sites for these proteins. Here we demonstrate that Drosophila TAF1 protein isoforms TAF1-2 and TAF1-4 directly bind DNA independently of TAF2. DNA binding by TAF1 isoforms is mediated by cooperative interactions of two identical AT-hook motifs, one of which is encoded by an alternatively spliced exon. Electrophoretic mobility shift assays revealed that TAF1-2 bound the minor groove of adenine-thymine-rich DNA with a preference for the sequence AAT. Alanine-scanning mutagenesis of the alternatively spliced AT-hook indicated that Lys and Arg residues made essential DNA contacts, whereas Gly and Pro residues within the Arg-Gly-Arg-Pro core sequence were less important for DNA binding, suggesting that AT-hooks are more divergent than previously predicted. TAF1-2 bound with variable affinity to the transcription start site of several Drosophila genes, and binding to the hsp70 promoter was reduced by mutation of a single base pair at the transcription start site. Collectively, these data indicate that AT-hooks serve to anchor TAF1 isoforms to the minor groove of adenine-thymine-rich Drosophila gene promoters and suggest a model in which regulated expression of TAF1 isoforms by alternative splicing contributes to gene-specific transcription.Binding of core promoters by the general transcription factor TFIID 2 correlates with transcription activity for most RNA polymerase II genes (1-3). In metazoans, core promoters for RNA polymerase II genes comprise ϳ70 bp surrounding the transcription start site (TSS) and are made up of ϳ8-bp elements (4, 5). Core promoter elements include the TATA box located 26 -31 bp upstream of the TSS, the initiator element located at the TSS, and the downstream promoter element located 28 -33 bp downstream of the TSS. Multiple subunits of TFIID engage in DNA binding at the core promoter. TATA-binding protein binds the TATA box and TATA-binding protein-associated factors (TAFs) bind downstream elements (6, 7). Several lines of evidence point to the importance of TAF1-core promoter DNA interactions. Studies in yeast and mammalian cells indicate that core promoters, not activator-binding sites, render genes TAF1-dependent (8 -10); cross-linking studies in Drosophila place TAF1 in close proximity to the TSS of the hsp70 gene (11); studies with purified human TFIID reveal TAF1 as the major TAF species that can be cross-linked to the downstream core element of several promoters (12); and TAF1⅐TAF2 heterodimers preferentially bind initiator sequences in vitro (8,13,14). However, TAF1 domains required for core promoter interactions have not been ident...