The highly specific interaction of each aminoacyl-tRNA synthetase and its s
ubstrate tRNAs constitutes an intriguing problem in protein-RNA recognition
. All tRNAs have the same overall three-dimensional structure in order to f
it interchangeably into the translational apparatus. Thus, the recognition
by aminoacyl-tRNA synthetase must be more or less limited to discrimination
between bases at specific positions within the tRNA. The hypermodified nuc
leotide 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) present at the wobb
le position of bacterial tRNAs specific for glutamic acid, lysine and possi
bly glutamine has been shown to be important in the recognition of these tR
NAs by their synthetases in vitro. Here, we have determined the aminoacylat
ion level in vivo of tRNA(Glu), tRNA(Lys), and tRNA(1)(Gln) in Escherichia
coli strains containing undermodified derivatives of mnm(5)s(2)U34. Lack of
the 5-methylaminomethyl group did not reduce charging levels for any of th
e three tRNAs. Lack of the s(2)U34 modification caused a 40% reduction in t
he charging level of tRNA(Glu). Charging of tRNA(Lys) and tRNA(1)(Gln) were
less affected. There was no compensating regulation of expression of gluta
myl-tRNA synthetase because the relative synthesis rate was the same in the
wild-type and mutant strains. These results indicate that the mnm(5)U34 mo
dification is not an important recognition element in vivo for the glutamyl
-tRNA synthetase. In contrast, lack of the s(2)U34 modification reduced the
efficiency of charging by at least 40%. This is the minimal estimate becau
se the turn-over rate of Glu-tRNA(Glu) was also reduced in the absence of t
he 2-thio group. Lack of either modification did not affect mischarging or
mistranslation. (C) 1998 Academic Press.