Functional Anticodon Architecture of Human tRNALys3 Includes Disruption of Intraloop Hydrogen Bonding by the Naturally Occurring Amino Acid Modification, t6A

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• 作者：John W. Stuart ; Zofia Gdaniec ; Richard Guenther ; Michal Marszalek ; Elzbieta Sochacka ; Andrzej Malkiewicz ; and Paul F. Agris
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：November 7, 2000
• 年：2000
• 卷：39
• 期：44
• 页码：13396 - 13404
• 全文大小：508K
• 年卷期：v.39,no.44(November 7, 2000)
• ISSN：1520-4995

文摘

The structure of the human tRNA^Lys3 anticodon stem and loop domain (ASL^Lys3) providesevidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t⁶A₃₇) to tRNA^Lys3functions. The t⁶A₃₇-modified anticodon stem and loop domain of tRNA^Lys3_UUU (ASL^Lys3_UUU- t⁶A₃₇) witha UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL^Lys3_UUUis 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 thefirst reported of an RNA in which a naturally occurring hypermodified nucleoside was introduced byautomated chemical synthesis. The ASL^Lys3_UUU-t⁶A₃₇ loop is significantly different than that of theunmodified ASL^Lys3_UUU, although the five canonical base pairs of both ASL^Lys3_UUU stems are in the standardA-form of helical RNA. t⁶A₃₇, 3'-adjacent to the anticodon, adopts the form of a tricyclic nucleoside withan intraresidue H-bond and enhances base stacking on the 3'-side of the anticodon loop. Critically importantto ribosome binding, incorporation of the modification negates formation of an intraloop U₃₃·A₃₇ basepair that is observed in the unmodified ASL^Lys3_UUU. The anticodon wobble position U₃₄ nucleobase inASL^Lys3_UUU-t⁶A₃₇ is significantly displaced from its position in the unmodified ASL and directed awayfrom the codon-binding face of the loop resulting in only two anticodon bases for codon binding. Thisconformation is one explanation for ASL^Lys3_UUU tendency to prematurely terminate translation and -1frame shift. At the pH 5.6 conditions of our structure determination, A₃₈ is protonated and positivelycharged in ASL^Lys3_UUU-t⁶A₃₇ and the unmodified ASL^Lys3_UUU. The ionized carboxylic acid moiety of t⁶A₃₇possibly neutralizes the positive charge of A⁺₃₈. The protonated A⁺₃₈ can base pair with C₃₂, but t⁶A₃₇may weaken the interaction through steric interference. From these results, we conclude that ribosomebinding cannot simply be an induced fit of the anticodon stem and loop, otherwise the unmodifiedASL^Lys3_UUU would bind as well as ASL^Lys3_UUU-t⁶A₃₇. t⁶A₃₇ and other position 37 modifications producethe open, structured loop required for ribosomal binding.