Self-association of the Amino-terminal Domain of the Yeast TATA-binding Protein

Abstract

The amino-terminal domain of yeast TATA-binding protein has been proposed to play a crucial role in the self-association mechanism(s) of the full-length protein.Here we tested the ability of this domain to self-associate under a variety of solution conditions. Escherichia coli two-hybrid assays, in vitro pull-down assays, and in vitro cross-linking provided qualitative evidence for a limited and specific self-association. Sedimentation equilibrium analysis using purified protein was consistent with a monomer-dimer equilibrium with an apparent dissociation constant of ϳ8.4 M. Higher stoichiometry associations remain possible but could not be detected by any of these methods. These results demonstrate that the minimal structure necessary for aminoterminal domain self-association must be present even in the absence of carboxyl-terminal domain structures. On the basis of these results we propose that aminoterminal domain structures contribute to the oligomerization interface of the full-length yeast TATA-binding protein.The TATA-binding protein (TBP) 1 is required for transcription initiation in eukaryotes and Archaea (1, 2). Together with TBP-associated protein factors (TAFs (3, 4)), it forms multisubunit complexes required for transcription by RNA polymerases I, II, and III (2, 5-7). TBP is a two-domain protein with a strongly conserved carboxyl-terminal domain (ϳ80% sequence identity among eukaryotes (8, 9)) and an amino-terminal domain highly divergent in both length (18 -173 residues) and sequence (8, 10 -12). The carboxyl-terminal domain is required for DNA binding and interactions with TAFs, general transcription factors (2,13,14), and self-association reactions (15,16). Less is known about the function of the amino-terminal domain, although it has been shown to be necessary for activated transcription of some polymerase II-and III-dependent genes (8,10,(17)(18)(19)(20).TBP functions in transcription initiation as a monomer. It binds TATA sequence DNA as a monomer (21-26) and interacts with TAFs and general transcription factors as a monomer (13,14,(27)(28)(29). On the other hand, TBP is capable of selfassociation. Dimers, tetramers, octamers, and higher oligomers have been previously reported in vitro (15, 16, 30 -35), while in vivo cross-linking results are consistent with association to at least the dimer level in HeLa and yeast cells (16,36,37). On the basis of these results, it has been proposed that monomeroligomer equilibria affect the concentration of TBP monomer that is available for transport into the nucleus for transcription-regulatory functions or for degradation (15, 16, 32-34, 38, 39).Little is known about the mechanism(s) of TBP self-association. Crystal structures of the carboxyl-terminal domain of yeast TBP reveal a saddle-shaped molecule of ϳ180 amino acid residues with a concave DNA-binding face and a convex TAFand transcription factor-binding face (9,21,23,30,31). In the absence of DNA, this crystallizes as a dimer, stabilized by extensive contacts between the concave surfaces (9, 30, 31...

Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

“…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).…”

Stalking metal-linked dimers

“…6A-C). The assay has been successfully used to detect homodimerization in the eukaryotic TATA binding protein (TBP) involved in transcription of nuclear-encoded genes [50,51], among others [41]. Detection of association between unlike proteins has also been demonstrated [52].…”

Incidence of deformities and variation in shape of mentum and wing of Chironomus columbiensis (Diptera, Chironomidae) as tools to assess aquatic contamination

“…Different authors indicate that morphological variations in aquatic individuals can be an indication of contamination and, thereby, can be considered a potential tool to evaluate the quality of water sources [8, 9, 2, 6]. Within this context, it is important to have a precise method in the comparative analyses of the morphology of the organisms and their possible variations, as well as the geometric morphometrics that becomes a precise tool when quantifying the morphological variation of a structure [10, 11, 12, 13].…”