The modified wobble nucleoside uridine-5-oxyacetic acid in tRNA^Pro_cmo⁵UGG promotes reading of all four proline codons in vivo

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 (tRNA^Pro_cmo5UGG) 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 tRNA^Pro_cmo5UGG 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 tRNA^Pro_cmo5UGG 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^Pro_cmo5UGG 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.

Keywords

• tRNA
• modified nucleoside
• wobble
• family codon box
• uridine-5-oxyacetic acid
• synthesis