Functional anticodon architecture of human tRNA(Lys3) includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t(6)A

The structure of the human tRNA(Lys3) anticodon stem and loop domain (ASL(L ys3)) provides evidence of the physicochemical contributions of N6-threonyl carbamoyladenosine (t(6)A(37)) to tRNA(Lys3) functions. The t(6)A(37)-modif ied anticodon stem and loop domain of tRNA(UUU)(Lys3) (ASL(UUU)(Lys3)-t(6)A (37)) With a UUU anticodon is bound by the appropriately programmed ribosom es, but the unmodified ASL(UUU)(Lys3) is not [Yarian, C., Marszalek, M., So chacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390-13395]. The structure, determined to an average rms d of 1.57 +/- 0.33 Angstrom, (relative to the mean structure) by NMR spectr oscopy and restrained molecular dynamics, is the first reported of an RNA i n which a naturally occurring hypermodified nucleoside was introduced by au tomated chemical synthesis. The ASL(UUU)(Lys3)-t(6)A(37) loop is significan tly different than that of the unmodified ASL(UUU)(Lys3), although the five canonical base pairs of both ASL(UUU)(Lys3) stems are in the standard A-fo rm of helical RNA. t(6)A(37), 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base st acking on the 3'-side of the anticodon loop. Critically important to riboso me binding, incorporation of the modification negates formation of an intra loop U-33.A(37) base pair that is observed in the unmodified ASL(UUU)(Lys3) . The anticodon wobble position U-34 nucleobase in ASL(UUU)(Lys3)-t(6)A(37) is significantly displaced from its position in the unmodified ASL and dir ected away from the codon-binding face of the loop resulting in only two an ticodon bases for codon binding. This conformation is one explanation for A SL(UUU)(Lys3) tendency to prematurely terminate translation and -1 frame sh ift. At the pH 5.6 conditions of our structure determination, A(38) is prot onated and positively charged in ASL(UUU)(Lys3)-t(6)A(37) and the unmodifie d ASL(UUU)(Lys3). The ionized carboxylic acid moiety of t(6)A(37) possibly neutralizes the positive charge of A(38)(+). The protonated A(38)(+) can ba se pair with C-32, but t(6)A(37) may weaken the interaction through steric interference. From these results, we conclude that ribosome binding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmo dified ASL(UUU)(Lys3) would bind as well as ASL(UUU)(Lys3)-t(6)A(37). t(6)A (37) and other position 37 modifications produce the open, structured loop required for ribosomal binding.