Fig. 5.

DBA-linked Tsr2E64G mutant is impaired in binding ESS2. a hTsr2E64G mutant expressed in yeast impairs yeast growth. Overexpression of eS26 rescues from the impaired growth. The conditional PGAL1-TSR2 strain was co-transformed with 2μ vectors expressing indicated genes. Transformants were spotted in 10-fold dilutions on repressive glucose containing media and grown at indicated temperatures for 2–4 days. b Tsr2 DBA mutant cells (hTsr2E64G) accumulate immature 20S pre-rRNA in the cytoplasm. P_GAL1-TSR2 cells were grown at 30 °C in glucose containing media to mid-log phase. Localization of 20S pre-rRNA was analyzed by FISH using a Cy3-labeled oligonucleotide complementary to the 5′ portion of ITS1 (red). Nuclear and mitochondrial DNA was stained with DAPI (blue). Scale bar = 5 µm. c hTsr2E64G mutant inefficiently binds eS26. GST-hTsr2 and GST-hTsr2E64G were immobilized on Glutathione Sepharose before incubation with E. coli lysate containing recombinant eS26 and incubated at indicated salt concentrations. Bound proteins were eluted by SDS sample buffer, separated by SDS-PAGE, visualized by Coomassie Blue staining. L = input (1:10 diluted). The error bars show the standard deviation. d The far-UV CD spectra of both human WT Tsr2 and the E64G-DBA variant. The midpoints of the thermal denaturation curves followed at 222 nm are at 57 °C for both proteins. e Isothermal titration calorimetry (ITC) measurements of human Tsr2 with human ESS2. The binding isotherms were plotted against the molar ratio. The measured parameters and K_d values are indicated within the plots