Engineering regulated expression of a heterologous tRNACAGSer in yeast. A) The recombinant mistranslating tRNACAGSer gene (cloned into plasmid pRS305K-tetO-tRNA) was integrated into the yeast LEU2 locus by homologous recombination using the KanMX4 gene as a selectable marker. The same yeast strain was transformed with the pGalTRI plasmid containing the GAL1 promoter - tetR construct. Selection was carried out in MMgalactose-URA containing geneticin (200 mg/L). B) Growth curves of tetO-tRNA clones growing in liquid MMgalactose+geneticin at 30°C. Expression of the tRNACAGSer was induced by addition of 40 μg/mL of tetracycline at OD600 = 0.4 to 0.5 (T0'). Yeast growth was monitored by measuring OD600 of the culture or by counting the number of cells per mL using a Neubauer cell counting chamber. The dilution shown indicates start of second cultures where the tetracycline concentrations tested are indicated in the inset key in μg/mL. C) Left panel shows the amount of β-gal protein expressed in Control and mistranslating yeast cells at T90'. Center panel shows the residual activity of β-gal after its thermal inactivation at 47°C for 10 minutes. The activity of the β-gal fraction that remained functional after thermal inactivation and refolding (4°C) was determined by incubating cell extracts at 37°C for two minutes in the presence of ONPG. The values in the graph represent activity in tetO-tRNA cells as percent relative to Control cells. The right panel shows increased aggregation of mistranslated β-gal relative to wild type enzyme, confirming that mistranslation is an important source of protein aggregation. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between tetO-tRNA and Control cells in each graph are shown - *P < 0.05; **P < 0.01.
Paredes et al. BMC Biology 2012 10:55 doi:10.1186/1741-7007-10-55