title: A gain-of-function mutation in the second tetratricopeptide repeat of TFIIIC131 relieves autoinhibition of Brf1 binding.
abstract: 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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Robyn D
authors.initials: RD
authors.lastname: Moir
authors.forename: Karen V
authors.initials: KV
authors.lastname: Puglia
authors.forename: Ian M
authors.initials: IM
authors.lastname: Willis
mesh_terms: D019943:Amino Acid Substitution; D004271:DNA, Fungal / Q000378:metabolism; D005799:Genes, Dominant; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012320:RNA Polymerase III / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000378:metabolism*; D019474:Transcription Factors, TFIII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000098832:Transcription Factor TFIIIC
chemical_list: C090956:BRF1 protein, S cerevisiae; D004271:DNA, Fungal; D008024:Ligands; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III