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.
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: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, 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.
Madore
E
E
Départament de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada.
Florentz
C
C
Giegé
R
R
Sekine
S
S
Yokoyama
S
S
Lapointe
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005975:Glutamate-tRNA Ligase / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011560:Pseudouridine / Q000737:chemistry; D012352:RNA, Transfer, Glu / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D012352:RNA, Transfer, Glu; D011560:Pseudouridine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D005975:Glutamate-tRNA Ligase