Modified wobble uridines present in yeast tRNA reading twofold degenerate codon boxes (Fig. 2). 5-methylcarboxymethyl-2-thiouridine (mcm⁵s²U), 5-methylcarboxymethyl-uridine (mcm⁵U), and 5-carbamoylmethyl-2′-O-methyluridine (ncm⁵Um).

Sequence alignment of four TtcA and four Tuc1p homologs and six MnmA/Mtu1p proteins. Letters shaded black, dark-blue, and light-blue indicate that the indicated amino acid is found in this position in at least 100%, 55%, and 25% of the sequences in the alignment, respectively. Letters shaded red indicate conserved cysteine residues. Cys-X₁-X₂ -Cys motifs in the central domain of Groups 1 and 2 and in the flanking regions in Group 2 are, consequently, seen as two red stripes close to each other. Conserved regions thought to be important for binding and catalytic function are boxed in red. Amino acids identified by Numata et al. (2006) important for binding of the tRNA substrate (black dots) and for catalytic function (open circles), as well as the two sulphur coordinating cysteines (C102 and C199 in E. coli MnmA, red dots) are indicated. At bottom is shown a schematic comparison of Group 1 (TtcA) and Group 2 (Ygl211p, renamed to Tuc1p, see text) as suggested by Jäger et al. (2004). The central part of MnmA, which is required for the synthesis of s²U34 in bacteria, is distinctly different from Groups 1 and 2 and is similar to the yeast protein Mtu1p, which is responsible for s²U34 formation in mitochondria (Umeda et al. 2005). The central domains of both Group 1 and Group 2 consist of the PP-loop (SGGxDS) motif, a Cys-X₁-X₂-Cys motif, a GH motif, and a PL motif. In addition to the conserved central domain, Group 2 proteins have two Cys-X₁-X₂-Cys motifs on each side of the central domain. Note that MnmA has the PP-loop motif and GH motif, but otherwise is not similar to any of the TtcA family proteins, but instead has conserved regions absent in Groups 1/2 thiolases.

HPLC analysis of modified nucleosides of tRNA from W303-1B (wild-type; thick line) and from strain UMY3400 (elp3∷KanMX4 tuc1∷TRP/pRS425-tE[UUC]-tK[UUU]-tQ[UUG]) (tE, tK and tQ indicate the genes for tRNA^Glu _mcm5s2UUC, tRNA^Lys _mcm5s2UUU, and tRNA^Gln _mcm5s2UUG; thin line). Transfer RNA was degraded to nucleosides and the distribution of nucleosides was analyzed by HPLC. (A–C) The 13–19 min region, 37–41 min region, and 49.5–54 min region, respectively, of the HPLC chromatogram monitored at 254 nm. (D) The region 30–58 min was monitored at 314 nm. The expected position of s²U is indicated at bottom, and this compound was observed only in tRNA from UMY2843 (elp3∷KanMX4). The identities of cm⁵s²U and ncm⁵s²UmpA are tentative, since no markers for these compounds were available.

Modified nucleosides in the wobble position and the coding capacities of the corresponding tRNAs. Letters outside the box, to the left, at top, and to the right indicate the first, second, and third position of the codon, respectively. Circles connected by a line, or a single circle, represent one tRNA species. A filled circle indicates the capability of that tRNA to base pair with a particular codon, either by Watson–Crick or by wobble according to the current wobble rules (Yokoyama and Nishimura 1995). An open circle suggests a restricted base pairing. To the right of each tRNA, the modification at positions 34 (the wobble position) is shown. Data compiled from Johansson and Byström (2005). Thus, tRNA specific for Lys, having mcm⁵s²U as wobble nucleoside, pairs well with AAA (•) but less well with AAG (○). (A) Presence of mcm⁵s²U in tRNA^Gln and mcm⁵U in tRNA^Gly as wobble nucleosides has been determined (Lu et al. 2005) (M.J.O. Johansson, pers. comm.) (B) tRNAs specific for Ser, Thr, and Ala have ncm⁵U, which synthesis is dependent on ELP3 (M.J.O. Johansson, pers. comm.).

Increased expression of tRNAs normally having mcm⁵s²U suppresses lethality induced by lack of this modification of wobble uridines. (A) Strains containing the elp3∷KanMX4 tuc1∷TRP1 mutations and carrying the URA3 plasmid pRS316-ELP3 and the LEU2 plasmids pRS425, pRS315-ELP3, pRS-425-tE(UUC), pRS425-tQ(UUG), pRS425-tK(UUU), pRS425-tK(UUU)-tQ(UUG), pRS425-tK(UUU)-tE(UUC), or pRS425-tE(UUC)-tK(UUU)-tQ(UUG) (tE, tK and tQ indicate the genes for tRNA^Glu _mcm5s2UUC, tRNA^Lys _mcm5s2UUU, and tRNA^Gln _mcm5s2UUG, respectively), were grown in synthetic complete medium lacking leucine (SC-leu). Strains were diluted to 3 × 10⁷ cells/mL, 10-fold serially diluted, and 5 μL was spotted onto either SC-leu plates or SC-leu plates containing 5-fluoroorotic acid (5-FOA). These plates were incubated at 30°C for 2 and 3 d, respectively. Cells containing a URA3 plasmid are unable to grow on 5-FOA-containing medium, with the result that these strains only contain the indicated LEU2 vectors when plated on 5-FOA plates (Boeke et al. 1984). (B) Growth of wild-type (W303-1B) and of the double mutant elp3∷KanMX4 tuc1∷TRP1 containing plasmids harboring genes encoding various tRNAs on SC-plates lacking leucine. Cells were picked from SC-Leu+5-FOA plates (Fig. 4A) and restreaked on SC plates lacking Leu. Plates were incubated at 30°C or at 38°C for 2.5 d. Plasmid pRS315 and pRS425 are denoted lc (low copy) and hc (high copy), respectively. (C) Growth of wild-type (W303–1B), tuc1∷TRP1 (YMP006), elp3∷KanMX4 (UMY2843), or the double mutant tuc1∷TRP1, elp3∷KanMX4/ pRS425-tEKQ (UMY3400) on rich medium (YEPD). Plasmid pRS425-tEKQ is denoted hc-tEKQ and its presence results in overexpression of tRNA^Glu _mcm5s2UUC, tRNA^Lys _mcm5s2UUU, and tRNA^Gln _mcm5s2UUG. Growth at 30°C was scored after 1 or 2 d.

Levels of tRNA^Glu _mcm5s2UUC, tRNA^Gln _mcm5s2UUG, and tRNA^Lys _mcm5s2UUU in strains W303-1B (wt), UMY3400 (elp3∷KanMX4 tuc1∷TRP1/pRS425-tE(UUC)-tK(UUU)-tQ(UUG)), and UMY3463 (ELP3 TUC1/pRS425-tE(UUC)-tK(UUU)-tQ(UUG)). The tRNA levels were monitored by Northern hybridization using radioactive oligonucleotides complementary to nucleotides 57–72 of tRNA^Gln, 57–73 of tRNA^Lys, 52–72 of tRNA^Glu, and 30–49 of tRNA^Met. Use of the last oligonucleotide, which monitors the levels of a tRNA not having mcm⁵s²U, served as controls for how much material was loaded on the gel. The numbers below each lane represent the levels relative to the level of the corresponding tRNAs in strain W303-1B (wild type).