The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13 degrees bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three alpha-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel beta-structure and random coil that interacts with the minor groove.

The crystal structure of the transcription factor IIB (TFIIB)/TATA box-binding protein (TBP)/TATA-element ternary complex is described at 2.7 A resolution. Core TFIIB resembles cyclin A, and recognizes the preformed TBP-DNA complex through protein-protein and protein-DNA interactions. The amino-terminal domain of core TFIIB forms the downstream surface of the ternary complex, where it could fix the transcription start site. The remaining surfaces of TBP and the TFIIB can interact with TBP-associated factors, other class II initiation factors, and transcriptional activators and coactivators.

The TATA box-binding protein (TBP) is required by all three eukaryotic RNA polymerases for correct initiation of transcription of ribosomal, messenger, small nuclear, and transfer RNAs. The cocrystal structure of the C-terminal/core region of human TBP complexed with the TATA element of the adenovirus major late promoter has been determined at 1.9 A resolution. Structural and functional analyses of the protein-DNA complex are presented, with a detailed comparison to our 1.9-A-resolution structure ofArabidopsis thaliana TBP2 bound to the same TATA box.Eukaryotes have three distinct RNA polymerases (forms I, II, and III) that catalyze transcription of nuclear genes (1). Despite their structural complexity, these multisubunit enzymes require sets of auxiliary proteins known as general transcription initiation factors to initiate transcription from corresponding class I, II, and III nuclear gene promoters (2)(3)(4)(5) We now report the crystal structure of human C-terminal/ core TBP (hTBPc) recognizing the AdMLP TATA element. The 1.9-A-resolution structure permits detailed structural and functional analyses of the protein-DNA interactions and provides further insights into how TBPs from different organisms recognize and direct transcription initiation from TATAcontaining promoters. MATERIALS AND METHODSCrystallization. hTBPc residues 155-335 (15) fused with an additional 20 N-terminal amino acids (MGSSHHHHHHSS-GLVPRGSH) was overexpressed in Escherichia coli [BL21-(DE3)pLysS] using the T7 RNA polymerase system (16). Cells grown at 30°C to an absorbance of 0.7 at 595 nm and induced with 0.5 mM isopropyl P3-D-thiogalactopyranoside for 3 h were harvested by low-speed centrifugation. Lysis was performed by three cycles of freeze-thaw and DNA digestion was performed by DNase I. The soluble fraction was applied directly to a Ni2+ ion affinity column and washed with a buffer containing increasing amounts of imidazole. hTBPc with an estimated homogeneity of 95% was then eluted from the resin with a buffer containing 100 mM EDTA. After overnight dialysis, removal of the histidine-containing N-terminal sequence was Abbreviations: AdMLP, adenovirus major late promoter; PIC, preinitiation complex; TBP, TATA box-binding protein; hTBPc, human core TATA box-binding protein; TBP2, TATA box-binding protein isoform 2; pol II, RNA polymerase II; rmsd, rms deviation.Data deposition: The atomic coordinates and structure factor amplitudes have been deposited in the Protein