NMR studies of the effects of the 5′-phosphate group on conformational properties of 5-methylaminomethyluridine found in the first position of the anticodon of Escherichia coli tRNA^Arg₄

5-Methylaminomethyluridine (mnm⁵U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA^Arg₄ for codons AGA/AGG. In the present study, the temperature dependence of the ribose-puckering equilibrium of pmnm⁵U was analyzed by proton NMR spectroscopy. Thus, the enthalpy difference (ΔH) between the C2′-endo and C3′-endo forms was obtained as 0.65 kcal·mol^-1. By comparison of the ΔH values of pU and pmnm⁵U, the 5-substitution was found to increase the relative stability of the C3′-endo form over the C2′-endo form significantly (by 0.56 kcal·mol^-1). Furthermore, this conformational "rigidity" was concluded to depend on the 5′-phosphate group, because nucleoside U exhibits only a negligible change in the ribose-puckering equilibrium upon the 5-methylaminomethyl substitution. Further NMR analyses and molecular dynamics calculations revealed that interactions between the 5-methylaminomethyl and 5′-phosphate groups of pmnm⁵U restrict the conformation about the glycosidic bond to a low anti form, enhancing steric repulsion between the 2-carbonyl and 2′-hydroxyl groups in the C2′-endo form. This intrinsic conformational rigidity of the mnm⁵U residue in position 34 may contribute to the correct codon recognition.