Figure 4.

Evidence that newly synthesized initiator tRNA^Met is unstable in gcd10 mutants. (A) Transformants of strain H2457 (gcd10-504) bearing the GCD10 plasmid pMG107 (GCD10) or vector YEp24 (gcd10-504) were grown in supplemented SD medium at 36°C for 2.25 hr before the addition of 5.0 mCi [5,6-3H]-uracil (37 Ci/mmole, 1 mCi/ml NEN). Cells were continuously labeled at 36°C for 60 min (pulse) after which 200-fold excess unlabeled uracil was added and incubation at 36°C was continued for 5 hr (chase). Total RNA was isolated from 2.0-ml aliquots at 0, 1, 3, and 5 hr after addition of unlabeled uracil and an amount of RNA representing equal cpms was hybridized to membrane-bound oligonucleotides complementary to full-length tRNA_i^Met (top) and tRNA_e^Met (bottom) in hybridization solution [500 mm NaCl, 24 mm NaH₂PO₄, 2.4 mm EDTA (pH 7.4), 30 % formamide, 5× Denhardt’s solution, 0.1% SDS] at 40°C for 2.5 days with constant mixing. After hybridization, filters were washed once in hybridization solution at 40°C for 30 min, once in 2× SSC, 0.1% SDS for 30 min at room temperature, once in 2× SSC for 30 min, and twice with 95% ethanol. Filters were dried and counted by liquid scintillation in Econo-fluor (Packard Chemical). The cpm bound to the membranes at each time point were corrected by subtracting the cpm bound to a third membrane containing a nonspecific oligonucleotide. The corrected counts per minute are expressed as the percentage of cpm bound to the membrane at the beginning of the chase (time = 0). (B) Strains YJA146 (gcd10Δ + hcIMT4) and a transformant of H2457 containing vector YEp24 (gcd10-504 + YEp24) were grown to mid-exponential phase at 26°C in SC or minimally supplemented SD medium and resuspended in the same medium prewarmed to 36°C containing 5 μg/ml thiolutin in DMSO or DMSO only. Northern blots of total RNA (10 μg) isolated from the strains at 26°C (0 hr at 36°C) or 36°C (4, 8, 12 hr at 36°C) with (+) or without (−) thiolutin treatment were probed with a labeled oligonucleotide that specifically hybridized to both pre-tRNA_i^Met and mature tRNA_i^Met, as described in Fig. 1. Labeled at right are various tRNA_i^Met species described in Figs. 2 and 3.