FIG. 1.

Cfd1 is required for 2-thio modification of cy-tRNAs but not of mt-tRNAs. (A) Total tRNAs prepared from TH-CFD1 cells grown without (−) or with (+) doxycycline (Dox) for 24 h (left panels) and from Gal-NFS1/YN cells grown with galactose for 20 h (Gal) or glycerol for 48 h (Gly) (right panels) were used. Isolated tRNAs (0.05 OD₂₆₀ unit [2 μg]) were loaded onto APM-containing (top panels) and APM-lacking (middle panels) denaturing gels, and after electrophoresis they were blotted to nylon membranes. These blots were hybridized with oligonucleotide probes specific to cy-tRNA_UUU^Lys2(cyK), cy-tRNA_UUC^Glu3(cyE), mt-tRNA_UCU^Arg(cyR), mt-tRNA_UUU^Lys(mtK), and mt-tRNA_UUG^Gln(mtQ). Positions of 2-thio-modified tRNAs that exhibited mobility retardation are indicated by a bracket, and positions of non-thio-modified tRNAs are indicated by an arrowhead. The protein levels of Cfd1 and Nfs1 were examined at time points similar to those described above by immunoblotting (bottom panels). (B) The relative proportions of 2-thio-modified tRNA in each tRNA were estimated. After the APM-containing gel electrophoresis shown in panel A, radioactivity bound to either the 2-thio-modified form or the unmodified form was measured as described in Materials and Methods. Values are represented as percentages of the amount of 2-thio-modified tRNA and the total amount of each tRNA (i.e., both the 2-thio modified and unmodified tRNA).