Eleven S. cerevisiae tRNA species contain an xm⁵U derivative at the wobble position. (A) Structures of mcm⁵U, mcm⁵s²U, ncm⁵U, and ncm⁵Um. (B) The indicated tRNA species was isolated from either wild-type (UMY2893) or elp3Δ (UMY2916) cells, and their nucleoside compositions were analyzed by HPLC. The part of the chromatogram between 10 and 20 min is shown in each case. The arrow indicates expected retention time of ncm⁵U (right panels). The small peak at this position represents an unrelated compound with an UV absorption spectrum different from that of ncm⁵U. (C) The genetic code and distribution of cytoplasmic S. cerevisiae tRNAs. The anticodon sequences of the 42 different tRNA species (1 initiator and 41 elongator tRNAs) are indicated (21, 25, 32, 39). For anticodons with an uncharacterized RNA sequence, the primary sequence is shown. The initiator and elongator tRNA^Met species have identical anticodon sequences. The wobble rules suggest that an inosine (I₃₄) residue allows paring with U, C, and sometimes A. A tRNA with a G or its 2′-O-methyl derivative (Gm) at the wobble position should read U- and C-ending codons. Presence of a C₃₄ residue or its 5-methyl (m⁵C) or 2′-O-methyl (Cm) variant should only allow pairing with G. The pseudouridine (Ψ)-containing tRNA^Ile is presumably unable to pair with the methionine AUG codon. The anticodons containing an xm⁵U derivative are shown in bold. In tRNA_A66^Pro, the A₃₄ residue is most likely modified to I₃₄ (see text). AU, absorbance units; wt, wild type.

Hypomodification induced by elp3Δ, tuc1Δ, or trm9Δ alleles does not reduce abundance or aminoacylation of any of the affected tRNA species. Northern blot analyses of total RNA isolated under either basic (A) or acidic (B) conditions from wild-type (W303-1B), elp3Δ (UMY2843), tuc1Δ (UMY3165), or trm9Δ (UMY3297) cells. After polyacrylamide gel electrophoresis and transfer under appropriate conditions, blots were probed for the U₃₄-containing tRNA of interest using ³²P-labeled oligonucleotides. To control for loading and indirect effects on aminoacylation, the blots were also probed for the C₃₄-containing initiator methionine tRNA (tRNA_i^Met). (A) Steady-state tRNA levels. (B) Aminoacylation levels. The first lane in each individual blot in panel B represents a deacylated wild-type RNA sample. The symbols for the U₃₄-containing tRNAs indicate the anticodon sequence of wild-type cells. The elp3Δ strain lacks the mcm⁵ and ncm⁵ side chains whereas the tuc1Δ strain lacks the s² groups. Cells deleted of TRM9 lack the esterified methyl group of mcm⁵ side chains. wt, wild type.

The wobble mcm⁵ side chain in tRNA_mcm⁵UCU^Argand tRNA_mcm⁵UCC^Glyimproves reading of G-ending codons. (A) Wild-type (W303-1B), elp3Δ (UMY2843), tr(ccu)jΔ (UMY3137), and elp3Δ tr(ccu)jΔ (UMY3136) cells carrying the low-copy-number URA3 plasmid pRS316-tR(CCU)J 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 for 3 days at 30°C. On 5-FOA-containing medium, only the cells that lost the URA3 plasmid were able to grow (5). The tR(CCU)J gene codes for tRNA_CCU^Arg. (B) The wild-type (W303-1B), elp3Δ (UMY2843), tg(ccc)dΔ tg(ccc)oΔ (UMY3304), and tg(ccc)dΔ tg(ccc)oΔ elp3Δ (UMY3320) strains were streaked on a YEPD (yeast extract, peptone, dextrose) plate and incubated at 30°C for 2 days. The tG(CCC) genes code for tRNA_CCC^Gly. wt, wild type.

Neither the mcm⁵ nor the s² group restricts tRNA_mcm⁵s²UUG^Glnand tRNA_mcm⁵s²UUC^Gluto A-ending codons. (A) The appropriate strains (W303-1B, UMY2843, UMY3165, UMY3133, UMY3134, UMY3347, UMY3348, UMY3350, and UMY3352) 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 tQ(CUG) and tE(CUC) genes code for tRNA_CUG^Glnand tRNA_CUC^Glu, respectively. (B) The tq(cug)Δ, te(cuc)Δ, and their elp3Δ or tuc1Δ 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 [tQ(UUG) or tE(UUC)]. 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. wt, wild type.

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 tRNA_CAC^Val, tRNA_CGA^Ser, and tRNA_CGU^Thr, 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 tRNA_AGG^Pro. wt, wild type.

Influence of the esterified methyl group of mcm⁵ side-chains on the decoding properties of tRNA. (A) The indicated strains (W303-1B, UMY3297, UMY3137, UMY3358, UMY3304, and UMY3360) were streaked on a YEPD (yeast extract, peptone, dextrose) plate and incubated at 30°C for 2 days. (B) The indicated strains (UMY3348, UMY3354, UMY3134, or UMY3345) 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 [tE(UUC) or tQ(UUG)]. The transformants were grown in SC-Leu medium for 24 h, serially diluted, spotted onto SC-Leu plates and SC-Leu plates containing 5-fluoroorotic acid (5-FOA), and incubated at 30°C for 2 days. wt, wild type.

The genetic code and decoding abilities of individual tRNA species. Codons read by a tRNA are indicated by circles and connecting lines. Red and pink circles represent tRNA species for which the decoding properties were investigated in this article. Pink circles connected with a dashed line indicate that the tRNA species reads the codon only when it is overexpressed. The empty dashed circle for tRNA_IAC^Valis shown only to indicate that this inosine-containing tRNA species does not efficiently read the GUA codon. The nucleoside at the wobble position is given for the 13 wobble uridine-containing tRNA species. Black and gray circles represent decoding abilities predicted by the wobble hypothesis, the revised wobble rules, and the distribution of tRNA species. A gray circle indicates that the tRNA species is less likely to read the codon. The number of genes coding for a tRNA species is indicated next to the circle for the complementary codon. The following qualifications apply: a superscript a indicates that the gene(s) encoding the tRNA is nonessential; a superscript b indicates that the gene(s) encoding the tRNA is essential; where two values are given (superscript c) four genes code for tRNA_i^Met, and five code for tRNA_m^Met.