Effect of modified nucleotides on Escherichia coli tRNA(Glu) structure andon its aminoacylation by glutamyl-tRNA synthetase - Predominant and distinct roles of the mnm(5) and s(2) modifications of U34

Overproducing Escherichia coli tRNA(Glu) in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNA(Glu) modivariant in wild-type E. co li contains five modified nucleosides: Psi 13, mnm(5)s(2)U34, m(2)A37, T54 and Psi 55. Four other overproduced modivariants differ from it by, respect ively. either the presence of an additional Psi, or the presence of s(2)U34 , or the lack of A37 methylation combined with either s(2)U34 or U34. Chemi cal probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, t he modivariant with neither mnm(5)s(2)U34 nor m(2)A37 has additional pertur bations in the D- and T-arms and in the variable region. The lack of a 2-th io group in nucleoside 34, which is mnm(5)s(2)U in the predominant tRNA(Glu ) modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tR NA synthetase for tRNA(Glu) in the aminoacylation reaction, showing that th is thio group is the identity element in the modified wobble nucleotide of E. coli tRNA(Glu). The modified nucleosides content also influences the rec ognition of ATP and glutamate by this enzyme. and in this case also, the pr edominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNA(Glu) modiv ariants indicate that the modification system of tRNA(Glu) optimizes the st ability of tRNA(Glu) and its action as cofactor of the glutamyl-tRNA synthe tase for the recognition of glutamate and ATP.