Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein.

The initiation of gene transcription in eukaryotes requires assembly of a multiprotein pre-initiation complex (PIC)1 that recruits RNA polymerase to a promoter. The binding of the TATA binding protein (TBP) to a TATA box sequence is a key step in PIC assembly at many promoters (1). Crystallographic and solution studies have established the formation of a 1:1 complex of TBP and TATA box (e.g., refs 2–5). TBP consists of a 180-residue C-terminal domain (TBPc) that is ~80% identical in sequence among all eukaryotes and a N-terminal domain that varies in both length and sequence (2). The shortest N-terminal domain consists of four residues in Pyrococcus woesei TBP (6) with the 158 residues of human TBP being the longest (7). The crystal structures of yeast, human, Arabidopsis, and archael TBP molecules that have been determined either lack or have rudimentary N-terminal domains (6, 8–10). In contrast to the large DNA conformation changes observed upon TBP or TBPc binding (11), little change is seen in the crystal structures of the C-terminal domain (6, 8, 9, 12). Only a small reorientation of the two subdomains relative to the 2-fold axis is observed in a manner independent of the sequence variations of the bound DNA (2, 13). Studies of the intrinsic fluorescence of TBP showed that the C-terminal domain has the same compact and rigid structure in free monomers and octamers and monomers complexed with DNA (4). Left unresolved was reconciliation of the high average tyrosine anisotropy with the demonstration by molecular dynamics of substantial mobility of two of the six tyrosine residues within the C-terminal domain (4). Although cells are viable when TBPc replaces TBP, the N-terminal domains of yeast and human TBP function in autoinhibition of DNA binding, binding of other transcription factors (14–18), and mediating self-assembly of the protein. Human and yeast TBPc dimerize in solution with equilibrium association constants and dissociation rates that vary greatly with the experimental conditions (19, 20). In contrast, TBP self-associates to tetramers and octamers (21, 22). Self-association and DNA binding are linked processes; only monomeric TBP–TATA box complexes are observed. While octamers rapidly equilibrate during DNA binding (23), equilibration of dimers can be slow (19, 20). The role of the N-terminal domain in mediating TBP self-association is highlighted by the ability of the isolated domain to itself self-associate (24). Limited information about the structure of the N-terminal domain of TBP and its relationship to the C-terminal core is available. Fluorescence spectroscopy was used to show that the structure of the N-terminal domain is different in the TBP–DNA complex, TBP monomers, and TBP octamers (4, 22, 25). This conclusion is consistent with hydroxyl radical protein footprinting of TBP and its complex with DNA, indicating that the N-terminal domain binds to the saddle of the core domain and is displaced upon DNA binding (5). The work presented here continues our exploration of the interdomain interactions by extending the fluorescence analysis of TBP structure and by directly comparing solution structural properties of TBP and TBPc. This study shows that the N-terminal domain undergoes structural changes and is an active participant in binding of the TATA box by the conserved C-terminal core domain. Properties of the C-terminal domain affected by the attached N-terminal domain include its DNA binding affinity and structural dynamics. An unexpected result is that binding of both TBPc and TBP is stabilized by divalent cations, albeit by different degrees. In general, the binding of TBPc to the TATA box sequence is more hydrophobic than that of TBP. We explore the hypothesis that the N-terminal domain of TBP functions as a TAF by undergoing structural and functional transformations necessary for formation of the promoter pre-initiation complex.