The 2.1-A crystal structure of an archaeal preinitiation complex: TATA-box-binding protein/transcription factor (II)B core/TATA-box

Abstract

Archaea possess a basal transcriptional apparatus that resembles that of eukaryotes. Here we report the 2.1-A crystal structure of the archaeal transcription factor complex formed by the TATA-box-binding protein (TBP), the transcription factor IIB homolog, and a DNA target, all from the hyperthermophile Pyrococcus woesei. The overall fold of these two basal transcription factors is essentially the same as that of their eukaryotic counterparts. However, in comparison with the eukaryotic complexes, the archaeal TBP-DNA interface is more symmetrical, and in this structure the orientation of the preinitiation complex assembly on the promoter is inverted with respect to that seen in all crystal structures of comparable eukaryotic systems. This study of the structural details of an archaeal transcription factor complex presents the opportunity to examine the evolution of the basal eukaryotic transcriptional apparatus from a stereochemical viewpoint and to extend our understanding of the physical biochemistry of transcriptional initiation.

Figure 1

(a) Amino acid sequences for pwTBP and pwTFB_c. The direct repeats of pwTBP and pwTFB are aligned. pwTBP amino acids in green are conserved with Arabidopsis thaliana TBP (atTBP), and pwTFB_c residues in green are conserved with human TFIIB_c. Red symbols above and below the sequence indicate interactions seen in this structure (d for DNA contacts and ∗ for protein contacts). Black symbols indicate the corresponding interactions in the eukaryotic ternary structure (11). Black rectangles indicate α-helices in pwTFB_c. Contacting residues are defined as having a distance < 4.0 Å. (b) Comparison of the promoter fragment crystallized in this study to five different TATA boxes seen in other TBP complex structures: yeast TBP (yTBP)–DNA (15), A. thaliana TBP (atTBP)–DNA (16, 17), human TBP (hTBP)–DNA (18), atTBP–human TFIIB–DNA (11), and yeast TBP–TFIIA–DNA (19, 20). Sequences are centered on the pseudodyad axis of the TATA box, and the orientation is shown with a schematic representation of the N-terminal and C-terminal domains of TBP. (c) Protein–DNA interactions in this structure. pwTBP residues are red ovals, and pwTFB_c residues are green rectangles. The boxA/TATA element is shaded. Black bars indicate the end of the crystallization oligonucleotide. Crystal packing contacts between DNA molecules simulate a contiguous B form helix with a smaller than normal twist of 9° at the junction. This allows pwTFB to interact with phosphates of neighboring molecules in both directions. Residues involved in van der Waals interactions are shown near the region of DNA they contact. Lines indicate hydrogen bonds or salt bridge interactions. (d) Residues interacting at the pwTBP–pwTFB_c interface. pwTBP residues are red and pwTFB_c residues, blue. Double rectangles or ovals indicate a main-chain interaction.

Figure 2

Stereo pair of electron density of a sigma A weighted (26) 2|F_o| − |F_c| map, contoured at 2σ. pwTFB_c residues are magenta, pwTBP residues are white, DNA is yellow, and water molecules are orange spheres.

Figure 3

(a) Ribbons (28) drawing of the overall structure of the ternary complex. pwTBP is in red, the N-terminal domain of pwTFB_c is green, the C-terminal domain is yellow, and DNA is blue. (b) Stereo drawing of pwTFB_c, with the same orientation as in a.

Figure 4

Ribbons (28) drawing of the archaeal and eukaryotic ternary complexes, with B-form DNA extended by modeling in both directions outside of the oligonucleotides used in crystallizations. DNA is depicted as a space-filling model. Proteins are depicted as ribbons. The N-terminal sequence repeat of TBP is red and the C-terminal repeat is blue, and TFB_c and TFIIB_c are green.

Figure 5

(a) Superposition of the N-terminal cyclin A-like domains of pwTFB_c (blue) and human TFIIB_c (orange) in the eukaryotic ternary complex (14). pwTFB_c residues 108–197 were aligned with human TFIIB residues 113–202, giving an rms deviation of 1.1 Å for corresponding C^α atoms. In a different superposition (not shown), pwTFB residues 211–300 can be aligned with human TFIIB_c residues 214–303, giving an rms deviation of 1.2 Å for corresponding C^α atoms. (b) Comparison of the electrostatic potential surface of pwTBP with A. thaliana TBP (atTBP), and human TFIIB_c (hTFIIB_c) with pwTFB_c. Molecular surfaces and electrostatic potentials were rendered with grasp (30), calculated using 0.1 M salt, and −2kT (red) to +2kT (blue). Coordinates for the eukaryotic ternary complex were provided by S. K. Burley (Rockefeller University, New York).