Interdependent interactions between TFIIB, TATA binding protein, and DNA

Abstract

Temperature-sensitive mutants of TFIIB that are defective for essential interactions were isolated. One mutation (G204D) results in disruption of a protein-protein contact between TFIIB and TATA binding protein (TBP), while the other (K272I) disrupts an interaction between TFIIB and DNA. The TBP gene was mutagenized, and alleles that suppress the slow-growth phenotypes of the TFIIB mutants were isolated. TFIIB with the G204D mutation [TFIIB(G204D)] was suppressed by hydrophobic substitutions at lysine 239 of TBP. These changes led to increased affinity between TBP and TFIIB. TFIIB(K272I) was weakly suppressed by TBP mutants in which K239 was changed to hydrophobic residues. However, this mutant TFIIB was strongly suppressed by conservative substitutions in the DNA binding surface of TBP. Biochemical characterization showed that these TBP mutants had increased affinity for a TATA element. The TBPs with increased affinity could not suppress TFIIB(G204D), leading us to propose a two-step model for the interaction between TFIIB and the TBP-DNA complex.

FIG. 1.

Positions of TBP and TFIIB mutants discussed in this paper. (A) Cocrystal structure of TBP, TFIIB, and DNA (31), but note that the amino acid numbers in reference have been changed to those of yeast TBP and TFIIB. TBP is shown in pink, TFIIB is shown in light blue, and DNA is shown in gray. The top view is a perspective from upstream of the transcription complex. The bottom view is the same, except rotated 45° as indicated. TFIIB residue K272 (contactingthe DNA phosphate backbone) is shown in dark blue and G204 (contacting the TBP stirrup) is shown in green. TBP residue K239, which suppresses both TFIIB mutants, is shown in red. (B) TBP suppressor mutations on the DNA binding surface. Starting with the upper view in panel A, TFIIB was removed and the structure was rotated to show a view from the DNA perspective. For orientation, K239 is shown again in dark orange. T124 and T215 are shown in dark blue, V213 is shown in cyan, Q68 and Q158 are shown in yellow, S118 and S209 are shown in purple, and A100 and A101 are shown in green.

FIG. 2.

Suppression of TFIIB(G204D) by TBP(K239L). (A) Suppression in vivo. Low-copy plasmids carrying the indicated alleles of the TBP gene (SPT15) were transformed into yeast strain YSB289 that carries the TFIIB(G204D) allele. Transformants were streaked for single colonies on selective media and incubated for 3 days at 30°C. (B) Suppression in vitro. Native gel electrophoresis was carried out on binding reactions containing different combinations of TBP [wild type (WT), lanes 1 and 2, or TBP(K239W) mutant, lanes 3 and 4] and TFIIB [wild type (WT), lanes 1 and 3, or TFIIB(G204D) mutant, lanes 2 and 4].

FIG. 3.

Suppression of TFIIB(K272I) by TBP mutant alleles. (A) Suppression in vivo. Low-copy plasmids carrying the indicated alleles of the TBP gene (SPT15) were transformed into yeast strain YSB288 that carries the TFIIB(K272I) allele. Transformants were streaked for single colonies on selective media and incubated for 3 days at 30°C. (B) Suppression in vitro. Native gel electrophoresis was carried out on binding reactions containing different combinations of TBP [wild type (WT), lanes 1 and 2, or TBP(T124A) mutant, lanes 3 and 4] and TFIIB [wild type (WT), lanes 1 and 3, or TFIIB(K272I) mutant, lanes 2 and 4]. Similar results were obtained with the S209N, Q68W, and S118N mutations.

FIG. 4.

TBP suppressors of TFIIB(K272I) have increased DNA binding affinity. (A) Native gel electrophoresis of wild-type and mutant TBP. Thirty nanograms of each protein was incubated with an AdMLP probe. Duplicate lanes for each protein are shown. (B) K_d values for wild-type and mutant TBPs. Titrations of TBP binding to the AdMLP were assayed using native gel electrophoresis, and the K_ds were calculated as described in Materials and Methods. K_ds for Q68H and Q68W were not significantly different from that for wild-type TBP.