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TATA Binding Protein (TBP) was first isolated and purified from humans in 1996. It is composed of a single polypeptide chain and is capable of binding specifically to the TATA box of many promoters. TBP is a saddle-shaped protein with a DNA binding fold. The concave DNA binding surface mediates specific and non-specific contacts with the DNA through an antiparallel B-sheet.
TBP is fully functional in mutants lacking the N-terminus. The C-terminus contains two homologous repeats of 88 amino acids. It is likely that TBP originally evolved from a true dimer of identical chains. The two almost identical domains have assymetric surface patterns of AA side chains. TBP can thus bind DNA and other proteins directionally.
Primary Structure
: TBP consist of one monomeric subunit 185 residues long. To view the sequences, click here.Weight: 2075 kD
Secondary Structure
Two motifs at both ends of the TBP-DNA binding domain each contain an antiparallel-B sheet of five strands and two helices. These two motifs are joined together by a short loop to make a saddle-shaped, ten stranded B-sheet. The B-sheets are highly involved in the DNA binding site; however, no alpha helices are involved (very strange in eukaryotic transcription). Another important structural feature of TBP is the pair of stirrups, one on each side of the saddle.
Ramachandran Plot
The above plot of the phi/psi angles reveals that most of the amino acids in this enzyme are either in alpha helices (correlating to the red region labeled A) or in anti-parallel pleated sheets (red region labeled B).
Solvent Accessible Surface Area
Solvent accessible residues reside primarily on the underside of the protein and include the hydrophobic and small uncharged polar side-chains projecting from each of the B-strands.
In many protein/DNA complexes, the DNA duplex is relatively unperturbed, and sequence recognition occurs via a pattern of hydrogen bonds from AA side-chains to N and O atoms along the floor of the major groove. However, the TBP/DNA complex is very unusual and does not follow this traditional binding strategy.
van der Waals' Contacts
The interaction area between the underside of the TBP saddle and the minor groove of DNA is formed by two large, complimentary surfaces with no water molecules between them. The interface is primarily hydrophobic, with fifteen side chains of TBP interacting with the base edges of the DNA. These residues occur in pairs at the centers of the central six B-strands.
Phenylalanine's Contribution
Two pairs of phe residues, one in each structural domain of the TBP moleucle, kink the helix by wedging between the outermost base pairs of the TATA element. Side chains of Phe284 and Phe301 open up base pairs 1 and 2, and side chains Phe193 and Phe210 open base pairs 7 and 8, making extenxive van der Waals' contacts. This produces two sharp kinks in the helix, destacks the base pairs and pulls them slightly apart. Any loss of stacking energy is compensated by extensive van der Waals' interaction between bases and the phe rings.
Hydrogen Bonding
Only four hydrogen bonds are observed between the TBP side chains and base edges, a clear difference of the usual process of protein/DNA recognition. At the very center of the complex, Asn253 and Asn163 hydrogen bonds with DNA bases. In addition, Thr309 and Thr218 hydrogen bond to other bases in the TATA sequence.
Sugar Phosphate/Base Edge Interactions
Of the 14 phosphates in the TATA box, only 6 are contacted directly or indirectly by the protein to form 6 direct or water mediated hydrogen bonds. The seven lys side chains close to the phosphates do not make direct or indirect hydrogen bonds to the phosphates. Instead, the epsilon ammonium groups of these residues are constrained through hydrogen bonds to acceptors within the protein. This suggests that the attractive electrostatic interaction may be mediated through a delocalized charge potential.
The underside of the saddle is also electrostatically positively charged. This positive charge furthur promotes TBP/DNA complex formation.
In conclusion, one important factor that contributes to the strong affinity of TBP proteins for TATA boxes is the large hydrophobic interaction area between them. Major distortions of the DNA structure cause the DNA to present a wide and shallow minor groove surface that is sterically complimentary to the underside of the saddle structure of the TBP protein. The complimentarity of these surfaces, and in addition the specific hydrogen bonds between side chains of TBP and bases in the minor groove, are the main factors responsible for causing TBP to bind to TATA boxes 100,000-fold more readily than to a random DNA sequence.
For more information on TBP, check out these great links at the Protein Data Bank.
References
1. Branden, Carl and John Tooze. Introduction to Protein Structure, Second Edition. Garland Publishing, 1999: New York.
2. Juo, Zong Sean, et al. "How Proteins Recognize the TATA Box." Journal of Molecular Biology. 1996 261: 239-254.
3. Kim, JL, Dimitar B. Nikolov, and Stephen K. Burley. "Co-crystal structure of TBP recognizing the minor groove of a TATA element." Nature. 1993 October 7; 365:520-527.
4. Kim, Youngchang, James Geiger, Steven Hahn, and Paul B. Sigler. "Crystal structure of a yeast TBP/TATA-box complex." Nature. 1993 October 7; 365:512-517.
5. Nikolov, Dimitar B., et al. "Crystal Structure of TFIID TATA-box Binding Protein. Nature. 1992 November 5; 360: 40-46.