Figure 11.

Second site suppressors of tra1${}_{\mathit{Q3}}$. (A) Second site suppressors rescue slow growth of tra1${}_{\mathit{Q3}}$. Yeast strains CY4353 (TRA1), CY6582 (tra1${}_{\mathit{Q3}}$) and four independent suppressors that restore growth of tra1${}_{\mathit{Q3}}$ on media containing ethanol were grown to stationary phase in media lacking leucine to maintain YCplac111-YHR100C, diluted in 10-fold dilutions and spotted onto YPD or YPD containing 6% ethanol and grown at 30° for two days. (B) Intragenic suppressors were mapped onto the cryo-EM Tra1 structure (Diaz-Santin et al. 2017). The three initial arginine to glutamine mutations are highlighted in red. Residues that suppressed tra1${}_{\mathit{Q3}}$ are blue. (C) Second site suppressors restore nuclear localization of Tra1${}_{\text{Q}3}$. An N-terminal eGFP tag was integrated in front of four tra1${}_{\mathit{Q3}}$ suppressor alleles. Yeast strains CY5998 (eGFP-TRA1), CY7341 (eGFP-tra1${}_{\mathit{Q3}}$) and the GFP tagged suppressors were grown to stationary phase in media lacking uracil, diluted 1:20 and grown to mid-logarithmic phase and stained with DAPI. Cells were washed twice with water before imaging. (D) Second site suppressors restore transcription defects of tra1${}_{\mathit{Q3}}$. Strains BY4741 (TRA1), CY6635 (tra1${}_{\mathit{Q3}}$) and suppressor strains CY6909 (tra1${}_{\mathit{Q3}}$-D3379N), CY6902 (tra1${}_{\mathit{Q3}}$-D3460Y), and CY6940 (tra1${}_{\mathit{Q3}}$-N3459I) were transformed with SRE-lacZ on a LEU2 centromeric plasmid. Strains were grown to saturation in media lacking leucine before being diluted 1:10 in YPD with 6% ethanol grown for eight hours. β-galactosidase activity units are the average of three replicates with the SD shown by the error bars.