Portal de Periódicos da CAPES

Functional Anticodon Architecture of Human tRNA Lys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t 6 A

2000; American Chemical Society; Volume: 39; Issue: 44 Linguagem: Inglês

10.1021/bi0013039

1943-295X

John W. Stuart, Zofia Gdaniec, Richard Guenther, Michal Marszalek, Elżbieta Sochacka, Andrzej Małkiewicz, Paul F. Agris,

RNA Research and Splicing

The structure of the human tRNALys3 anticodon stem and loop domain (ASLLys3) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t6A37) to tRNALys3 functions. The t6A37-modified anticodon stem and loop domain of tRNALys3UUU (ASLLys3UUU- t6A37) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASLLys3UUU is not [Yarian, C., Marszalek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P. F., Biochemistry 39, 13390−13395]. The structure, determined to an average rmsd of 1.57 ± 0.33 Å (relative to the mean structure) by NMR spectroscopy and restrained molecular dynamics, is the first reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced by automated chemical synthesis. The ASLLys3UUU-t6A37 loop is significantly different than that of the unmodified ASLLys3UUU, although the five canonical base pairs of both ASLLys3UUU stems are in the standard A-form of helical RNA. t6A37, 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside with an intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U33·A37 base pair that is observed in the unmodified ASLLys3UUU. The anticodon wobble position U34 nucleobase in ASLLys3UUU-t6A37 is significantly displaced from its position in the unmodified ASL and directed away from the codon-binding face of the loop resulting in only two anticodon bases for codon binding. This conformation is one explanation for ASLLys3UUU tendency to prematurely terminate translation and −1 frame shift. At the pH 5.6 conditions of our structure determination, A38 is protonated and positively charged in ASLLys3UUU-t6A37 and the unmodified ASLLys3UUU. The ionized carboxylic acid moiety of t6A37 possibly neutralizes the positive charge of A+38. The protonated A+38 can base pair with C32, but t6A37 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 unmodified ASLLys3UUU would bind as well as ASLLys3UUU-t6A37. t6A37 and other position 37 modifications produce the open, structured loop required for ribosomal binding.