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. 2004 Oct;10(10):1662-73.
doi: 10.1261/rna.7106404.

The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo

S Joakim Nasvall  1 , Peng Chen, Glenn R Bjork
Affiliations

The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAPro(cmo5UGG) promotes reading of all four proline codons in vivo

S Joakim Nasvall et al. RNA. 2004 Oct.

Abstract

In Salmonella enterica serovar Typhimurium five of the eight family codon boxes are decoded by a tRNA having the modified nucleoside uridine-5-oxyacetic acid (cmo5U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNAProcmo5UGG) of the three tRNAs that reads the four proline codons has cmo5U34. According to theoretical predictions and several results obtained in vitro, cmo5U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo5U34 in tRNAProcmo5UGG in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis of cmo5U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho5U34) instead of cmo5U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo5U34) and ho5U34 in tRNA. The results suggest that the synthesis of cmo5U34 occurs as follows: U34 -->(?) ho5U -->(CmoB) mo5U -->(CmoA?) cmo5U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNAProcmo5UGG and thus lacked the other two proline-specific tRNAs normally present in the cell. From analysis of growth rates of various strains and of the frequency of +1 frameshifting at a CCC-U site we conclude: (1) unexpectedly, tRNAProcmo5UGG is able to read all four proline codons; (2) the presence of ho5U34 instead of cmo5U34 in this tRNA reduces the efficiency with which it reads all four codons; and (3) the fully modified nucleoside is especially important for reading proline codons ending with U or C.

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Figures

FIGURE 1.
FIGURE 1.
(A) The genetic code. The eight codon boxes with shaded background are the family codon boxes, containing four codons representing one amino acid (fourfold degenerate). The five family codon boxes in lighter shade contain tRNA having cmo5U as a wobble nucleoside. The codon boxes with white background are the mixed codon boxes. (B) The proline family codon box (CCN). proK, L, and M denote the genes encoding tRNAProCGG, tRNAProGGG and tRNAProcmo5UGG, respectively, and the wobble nucleosides, which are present in position 34, are indicated. A circle corresponds to a codon read by a tRNA and a line connecting two or more circles indicates that the same tRNA is able to read those codons (e.g., the proL tRNA contains G34 and reads the CCU and CCC codons). The gray circle for tRNAProcmo5UGG (codon CCC) indicates that this tRNA reads the CCC codon (results presented in this article), and the black circles show the codon reading abilities predicted by the wobble hypothesis and the revised wobble rules.
FIGURE 2.
FIGURE 2.
The proposed biosynthetic pathway for the synthesis of cmo5U and mcmo5U. The dashed reaction arrows indicate the link between the synthesis of cmo5U and chorismic acid. The arrow denoted A is according to Hagervall et al. (1990) and the arrows denoted B and C are the suggested link between chorismic acid and the synthesis of cmo5U34 according to present results. (X?) A possible unknown derivative of chorismic acid. (U) Uridine, (ho5U) 5-hydroxyuridine, (mo5U) 5-methoxyuridine (cmo5U) uridine-5-oxyacetic acid, (mcmo5U) uridine-5-oxyacetic acid methyl ester.
FIGURE 3.
FIGURE 3.
3-Galactosidase assay showing suppression of the +1 frameshift mutation in lacZ of pTHF14. GT6631 is a mutant of the parental strain GT6606 that grew poorly on plates containing histidi-nol, but grew normally on plates containing histidine. All values are averages of three independent cultures and relative to the values in strain GT6606 (set to 1.0, which equals 1345 ± 88 miller units). A Student’s t-test (two-tailed; two sample equal variance) shows that both GT6607 and GT6631 are significantly lower than GT6606 (p < 0.001).
FIGURE 4.
FIGURE 4.
The gene organization of the cmoAB operon. (Top) The translational overlap between cmoA and cmoB is indicated with the RNA sequence and the corresponding peptide sequences. The start (AUG) codon for cmoB and the stop (UGA) codon for cmoA are indicated in bold. (Bottom) Schematic representation of the different mutants constructed in this study. The transposon EZ::TN 〈R6Kγori/KAN-2〉 is abbreviated EZ, the white triangle indicates the insertion point within the first few codons of the cmoA gene; kan is the kana-mycin resistance cassette from plasmid pKD4.
FIGURE 5.
FIGURE 5.
HPLC chromatograms monitored at 290 nm. The positions of cmo5U, ho5U, and mo5U are indicated above the corresponding peaks. The three major nucleosides C, U, and G are indicated below the corresponding peaks. “x” indicates an unidentified compound with a spectrum that is not similar to ho5U, comigrating with ho5U in all analyses. About 25 μg of total tRNA was digested to nucleosides and analyzed. The degradation procedure used, the treatment by alkaline phosphatase at pH 8.3, hydrolyzes most of mcmo5U to cmo5U.
FIGURE 6.
FIGURE 6.
Relative frameshifting frequencies at the CCC-UGA frameshifting site in hisD3749. All strains are ΔproL, hisO1242, hisD3749, hisD2504::MudK, and the values are relative to the corresponding in-frame construct (same genotype except without hisD3749) and are averages of three cultures. The cells were grown in LB + 100 mg/L kanamycin at 37°C. A Student’s t-test (two-tailed; two sample equal variance) shows that cmoA1<>frt, cmoB2<>frt, and cmoAB3<>frt significantly differ from GT6850 (wild type; p ≤ 0.001). cmoA1<>frt is also different from cmoB2<>frt and cmoAB3<>frt (p< 0.05).
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