5-Methylaminomethyluridine (mnm5U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA4Arg 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 (delta H) between the C2'-endo and C3'-endo forms was obtained at 0.65 kcal.mol-1. By comparison of the delta H 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.