A Role for TBP Dimerization in Preventing Unregulated Gene Expression

Abstract

The recruitment of the TATA box-binding protein (TBP) to promoters in vivo is often rate limiting in gene expression. We present evidence that TBP negatively autoregulates its accessibility to promoter DNA in yeast through dimerization. The crystal structure of TBP dimers was used to design point mutations in the dimer interface. These mutants are impaired for dimerization in vitro, and in vivo they generate large increases in activator-independent gene expression. Overexpression of wild-type TBP suppresses these mutants, possibly by heterodimerizing with them. In addition to loss of autorepression, dimerization-defective TBPs are rapidly degraded in vivo. Direct detection of TBP dimers in vivo was achieved through chemical cross-linking. Taken together, the data suggest that TBP dimerization prevents unregulated gene expression and its own degradation.

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

“…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).…”

Engineering Dimer-stabilizing Mutations in the TATA-binding Protein

“…TBP dimers in vitro is that they are present at lower steadystate levels in vivo (8,15). For example, TBP(N69R) is present in yeast cells at levels that are only about 10% of wild type TBP.…”

“…We reasoned that changing one or the other to an acidic residue might generate a positive electrostatic interaction whose effect can be measured in vitro using dimerization and DNA binding assays. Previous in vivo data suggested that TBP normally resides as dimers only when not bound to DNA (8,15). Under such circumstances, enhanced dimer stability might have little impact on its already autorepressed state.…”

“…The structure of TBP dimers has been defined crystallographically and through biochemical analysis. Dimer instability caused by mutations along the crystallographic dimer interface correlate with transcriptional derepression in yeast cells (8,15), and this derepression occurs genome-wide at about 7% of all genes (16). Together, these findings indicated that TBP dimerization represents a physiologically important mechanism for auto-inhibiting its DNA binding activity.…”

“…Under such circumstances, enhanced dimer stability might have little impact on its already autorepressed state. Therefore, we utilized a previously characterized unstable dimer mutant N69R (8) and created additional mutations at Arg-98 and Arg-171 to assess whether such mutations might suppress phenotypes associated with the N69R mutation. Phenotypes include lowered steady-state protein levels of the mutants, transcriptional derepression of a basal promoter, and inhibition of cell growth (toxicity).…”