PUS3 is not required for accumulation or aminoacylation of the mutant tRNAArgCCG. (A) Wild-type cells (PUS3 TRR4), trr4-1 cells (PUS3 trr4-1), cells containing PUS3 under control of the GAL1 promoter (GAL–PUS3 TRR4), and trr4-1 cells containing PUS3 under control of the GAL1 promoter (GAL–PUS3 trr4-1) were grown at 25°C in galactose-containing media and switched to media containing glucose at time 0. At the indicated intervals, RNA was extracted and subjected to Northern analysis to detect tRNAArgCCG (top) and tRNAArgUCU (bottom). (B) At intervals after the switch to glucose media, RNA was extracted from the above cells under acidic conditions and fractionated in acidic acrylamide gels. The Northern blot was probed to detect tRNAArgCCG (top) and tRNAArgUCU (bottom). (Lane 1) Deacylated wild-type tRNA. As described in Chakshusmathi et al. (2003), charged and uncharged forms of wild-type tRNAArgCCG migrate slightly faster than these forms of trr4-1 tRNAArgCCG.
