Ψ13 formation in cytoplasmic tRNAAsp (A) and tRNAGlu (B) is abolished upon PUS7 gene disruption and restored by complementation with plasmid p413GalS-PUS7. Total RNA was extracted from the WT and mat-a ΔPUS7 strains and modified by CMCT, for 1, 10, and 20 min with (+) or without (−) subsequent alkaline treatment (OH−). A control experiment was performed in the absence of CMCT treatment. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stops, corresponding to residues Ψ13, are indicated by arrows. To verify the direct involvement of Pus7p in the U to Ψ conversion at position 13 of tRNAs, the ΔPUS7 strain was transformed with plasmid p413GalS-PUS7, bearing the wild-type (WT) or mutated (D256A) PUS7 gene. Total RNA was extracted from these two transformed strains, and tRNAAsp (A) and tRNAGlu (B) were analyzed by the CMCT/RT approach (shown in lanes 7, 8, 9, and 10 in A and B).