Effect of modified nucleotides on Escherichia coli tRNAᴳˡᵘ structure and on its aminoacylation by glutamyl‐tRNA synthetase: Predominant and distinct roles of the mnm⁵ and s² modifications of U34
1999
Madore, Eric | Florentz, Catherine | Giegé, R. (Richard) | Sekine, Shun‐ichi | Yokoyama, Shigeyuki | Lapointe, Jacques
Overproducing Escherichia coli tRNAᴳˡᵘ in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAᴳˡᵘ modivariant in wild‐type E. coli contains five modified nucleosides: Ψ13, mnm⁵s²U34, m²A37, T54 and Ψ55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Ψ, or the presence of s²U34, or the lack of A37 methylation combined with either s²U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm⁵s²U34 nor m²A37 has additional perturbations in the D‐ and T‐arms and in the variable region. The lack of a 2‐thio group in nucleoside 34, which is mnm⁵s²U in the predominant tRNAᴳˡᵘ modivariant, decreases by 520‐fold the specificity of E. coli glutamyl‐tRNA synthetase for tRNAᴳˡᵘ in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAᴳˡᵘ. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAᴳˡᵘ modivariants indicate that the modification system of tRNAᴳˡᵘ optimizes the stability of tRNAᴳˡᵘ and its action as cofactor of the glutamyl‐tRNA synthetase for the recognition of glutamate and ATP.
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