The modified wobble nucleoside uridine-5-oxyacetic acid in tRNAcmo5UGGPro promotes reading of all four proline codons in vivo

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 (cmo(5)U) as a wobble nucleoside present in position 34 of the tRNA. In the proline family codon box, one (tRNA(cmo5UGG)(Pro)) of the three tRNAs that reads the four proline codons has cmo(5)U34. According to theoretical predictions and several results obtained in vitro, cmo(5)U34 should base pair with A, G, and U in the third position of the codon but not with C. To analyze the function of cmo(5)U34 in tRNA(cmo5UGG)(Pro) in vivo, we first identified two genes (cmoA and cmoB) involved in the synthesis Of cmo(5)U34. The null mutation cmoB2 results in tRNA having 5-hydroxyuridine (ho(5)U34) instead of cmo(5)U34, whereas the null mutation cmoA1 results in the accumulation of 5-methoxyuridine (mo(5)U34) and ho(5)U34 in tRNA. The results suggest that the synthesis of cmo(5)U34 occurs as follows: U34 -->(?) o(5)U -->(CmoB) mo(5)U -->(CmoA?) cmo(5)U. We introduced the cmoA1 or the cmoB2 null mutations into a strain that only had tRNA(cmo5UGG)(Pro) 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, tRNA(cmo5UGG)(Pro) is able to read all four proline codons; (2) the presence of ho(5)U34 instead of cmo(5)U34 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.