Ortholog replacement reveals stable TFIIIC complexes are required for proper mitotic chromosome segregation
Transcription factors are speculated to play crucial roles in adaptive evolution. Here we investigated how essential transcription factors (eTFs) change using ortholog replacement assays. Several orthologous eTFs from other yeast species could not fully complement the mutants in Saccharomyces cerevisiae, suggesting that these eTFs have changed their functions or interactions to become incompatible. We further characterized TFIIIC, a protein complex assisting RNA polymerase III transcription, that exhibits complete or partial incompatibility in several subunits. In the orthologous Tfc7-replacement line, the binding of TFIIIC to tRNA genes is reduced, but the abundance of tRNAs is not severely affected. However, Tfc7-replacement cells often mis-segregate chromosomes during mitosis and their fitness is further reduced in the spindle checkpoint mutant. Chromatin immunoprecipitation experiments showed that unstable TFIIIC binding results in defective cohesion loading, leading to chromosome mis-segregation. Swapping the highly divergent C-terminal domain of Tfc7 orthologs rescues its interaction with Tfc1 and cell fitness, suggesting that incompatibility is caused by altered interactions between complex subunits. Our results reveal separatable essential functions of a well-studied protein complex. Overall design: ChIP-seq of Saccharomyces cerevisiae S288C TFIIIC complex subunit TFC3 in wild type like Sc-TFC7 and orthologous replacement line Kl-TFC7