Effect of modified nucleosides on the structure of the anticodon domain of Escherichia coli tRNALys

The hypermodified nucleosides 5-(methylaminomethyl)-2-thiouridine (mnm/5s/2U) and N-[(9-beta-D-Ribofuranosyl-9H-purin-6-yl)carbamoyl)]-O-tert-butyidimethylsilyl-1-threonine-trimethlsilylethyl ester (t/6A) were synthesized as their phosphoramidites. The protected phosphoramidites were incorporated into the 17-nucleotide anticodon stemloop hairpin of Escherichia coli transfer ribonucleicacid lysine (E. coli tRNA/Lys ) with only modest changes in the automated oligonucleotide synthesis protocol to accomodate the sulfur and amine side chain of mnm/5s/2U and the threonine side chain of t/6A. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution structures of an RNA oligonucleotide comprising the 17-nucleotide anticodon domain of native E. coli tRNA Lys containing the three modified nucleosides, mnm/5s/2 U34, t 6 A37 and psi-39. The structural effects of nucleoside modification on the anticodon domain were determined by comparison of the anticodon stem-loop (ASL) structures of tRNA Lys -mnm/5/s/2 U,psi-39; tRNA/Lys-t/6A37,psi-39; and tRNA/Lys-mnm/5s/2U34,t/6A37,psi-39. The hypermodified mnm/5s/2U nucleoside stabilizes the canonical U-turn between U33 and the wobble nucleoside and concomitantly stabilizes stacking within the UUU anticodon. A stable U-turn is clearly indicated by a 31\ P NMR resonance shifted by more than 1.0 ppm downfield from the typical A-helical range. Sequential NOE connectivity is observed between the anticodon nucleosides 34-36 indicating contiguous stacking within the anticodon and a break between 33 and 34 as expected for the turn. The bulky side chain of t/6A is pushed away from the 5 ' side of stem-loop hairpin on account of steric hindrance. This result in bringing the 35-p-36 towards U33, thus is stabilizing the mnm/5s/2U34-induced U-turn. Although there is still appreciable C2 ' -endo character for the anticodon residues, the NOEs show that the anticodon has adopted a structure more similar to that of tRNA/Phe compared to the unmodified tRNA/Lys hairpin. The results from NMR experiments and CD experiments indicate that there is no direct interaction between the side chains of t/6A37 and mnm/5s2U34. Contrary to earlier claims, the anticodon loop structure of native E. coli tRNA/Lys is not unusual.