Open AccessEffect of modified nucleotides on Escherichia coli tRNA Glu structure and on its aminoacylation by glutamyl‐tRNA synthetase
Author(s) -
Madore Eric,
Florentz Catherine,
Giegé Richard,
Sekine Shunichi,
Yokoyama Shigeyuki,
Lapointe Jacques
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00965.x
Subject(s) - aminoacylation , transfer rna , escherichia coli , nucleotide , biochemistry , biology , wobble base pair , nucleoside , enzyme , methylation , rna , dna , gene
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. coli contains five modified nucleosides: Ψ13, mnm 5 s 2 U34, m 2 A37, T54 and Ψ55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Ψ, or the presence of s 2 U34, or the lack of A37 methylation combined with either s 2 U34 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 mnm 5 s 2 U34 nor m 2 A37 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 mnm 5 s 2 U in the predominant tRNA Glu modivariant, decreases by 520‐fold the specificity of E. coli glutamyl‐tRNA synthetase for tRNA Glu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNA Glu . 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 tRNA Glu modivariants indicate that the modification system of tRNA Glu optimizes the stability of tRNA Glu and its action as cofactor of the glutamyl‐tRNA synthetase for the recognition of glutamate and ATP.