The modification of the wobble base of tRNA(Glu) modulates the translationrate of glutamic acid codons in vivo

In Escherichia coli, uridine in the wobble position of tRNA(Glu) and tRNA(L ys) is modified to mnm(5)s(2)U34. This modification is believed to restrict the base-pairing capability, i.e. to prevent misreading of near-cognate co dons and reduce the efficiency of cognate codon reading, especially of codo ns ending in G. We have determined the influence of the 5-methylaminomethyl and the 2-thio modifications of mnm(5)s(2)U34 in tRNA(Glu) on the translat ion rate of the glutamate codons GAA and GAG in vivo. In wild-type cells, G AG is translated slower (7.7 codons/second) and GAA faster (18 codons/secon d) than the average codon (13 codons/second). Surprisingly, tRNA(Glu) lacki ng the 5-methylaminomethyl group, thus containing s(2)U34, translated GAA t wofold faster (47 codons/second) and GAG four-fold slower (1.9 codons/secon d) than fully modified tRNA(Glu). In contrast, tRNA(Glu) that contains mnm( 5)U34 instead of mnm(5)s(2)U34 translated GAA fourfold slower (4.5 codons/s econd) and GAG only 20% slower (6.2 codons/second). Clearly, the 5-methylam inomethyl group of mnm(5)s(2)U34 facilitates base-pairing with G while decr easing base-pairing with A, resulting in rates of translation of GAG and GA A that approach that of the average codon. The 2-thio group increases the r ecognition of GAA and has only a minor effect on the decoding of GAG. Furth ermore, the 2-thio group is important for aminoacylation (see the accompany ing paper). These data imply that the function of mnm(5)s(2)U34 may be diff erent from what has been suggested previously. (C) 1998 Academic Press.