DNA binding properties of TAF1 isoforms with two AT-hooks.
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 TAF1.TAF2 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.
Metcalf
Chad E
CE
Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA.
Wassarman
David A
DA
D024761:AT-Hook Motifs / Q000235:genetics*; D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D020033:Protein Isoforms; D035381:TATA-Binding Protein Associated Factors; D035362:Transcription Factor TFIID; D004247:DNA; D051548:Histone Acetyltransferases; C069001:TATA-binding protein associated factor 250 kDa