A gain-of-function mutation in the second tetratricopeptide repeat of TFIIIC131 relieves autoinhibition of Brf1 binding

The interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC, TFIIIC131, and the TFIIB-related factor Brf1 represents a limiting step in the assembly of the RNA polymerase III (pol III) initiation factor TFIIIB. This assembly reaction is facilitated by dominant mutations that map in and around TPR2. Structural modeling of TPR1 to TPR3 from TFIIIC131 shows that one such mutation, PCF1-2, alters a residue in the ligand-binding groove of the TPR superhelix whereas another mutation, PCF1-1, changes a surface-accessible residue on the back side of the TPR superhelix. In this work, we show that the PCF1-1 mutation (H190Y) increases the binding affinity for Brf1, but does not affect the binding affinity for Bdp1, in the TFIIIC-dependent assembly of TFIIIB. Interestingly, binding studies with TFIIIC131 fragments indicate that Brf1 does not interact directly at the site of the PCF1-1 mutation. Rather, the data suggest that the mutation overcomes the previously documented autoinhibition of Brf1 binding. These findings together with the results from site-directed mutagenesis support the hypothesis that gain-of-function mutations at amino acid 190 in TPR2 stabilize an alternative conformation of TFIIIC131 that promotes its interaction with Brf1.     

Interdependent interactions between TFIIB,TATA binding protein,and DNA

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.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.