FIG. 5.
A wobble ncm5 side chain improves reading of codons ending with G but does not restrict the tRNA to purine-ending codons. (A) The appropriate strains (W303-1B, UMY2843, UMY3296, UMY3333, UMY3126, UMY3127, UMY3129, and UMY3130) carrying the indicated low-copy-number URA3 plasmid were grown in liquid SC medium for 24 h. The cells were serially diluted, spotted onto SC plates and SC plates containing 5-fluoroorotic acid (5-FOA), and incubated at 30°C for 3 days. The tV(CAC), tS(CGA), and tT(CGU) genes code for tRNACACVal, tRNACGASer, and tRNACGUThr, respectively. (B) The ts(cga)cΔ, tt(cgu)kΔ, and their elp3Δ derivatives from panel A were transformed with an empty high-copy-number (h.c.) LEU2 vector (10) or the same plasmid harboring a gene coding for the relevant U34-containing tRNA [tS(UGA) or tT(UGU)]. The transformants were grown in SC-Leu medium for 24 h, serially diluted, spotted onto SC-Leu plates and SC-Leu plates containing 5-FOA, and incubated at 30°C for 3 days. (C) The wild-type (W303-1B), elp3Δ (UMY2843), tp(agg)cΔ tp(agg)nΔ (UMY3343), and tp(agg)cΔ tp(agg)nΔ elp3Δ (UMY3368) strains were streaked on a YEPD (yeast extract, peptone, dextrose) plate and incubated at 30°C for 2 days. The tP(AGG) genes code for tRNAAGGPro. wt, wild type.