The structure of the human tRNA Lys3 anticodon stem and loop domain (ASL Lys3 ) provides evidence of the physicochemical contributions of N6-threonylcarbamoyladenosine (t 6 A 37 ) to tRNA Lys3 functions. The t 6 A 37 -modified anticodon stem and loop domain of tRNA Lys3 UUU (ASL Lys3 UUU -t 6 A 37 ) with a UUU anticodon is bound by the appropriately programmed ribosomes, but the unmodified ASL Lys3 UUU 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 ASL Lys3 UUU -t 6 A 37 loop is significantly different than that of the unmodified ASL Lys3 UUU , although the five canonical base pairs of both ASL Lys3 UUU stems are in the standard A-form 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 stacking on the 3′-side of the anticodon loop. Critically important to ribosome binding, incorporation of the modification negates formation of an intraloop U 33 ‚A 37 base pair that is observed in the unmodified ASL Lys3 UUU . The anticodon wobble position U 34 nucleobase in ASL Lys3 UUU -t 6 A 37 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 ASL Lys3 UUU tendency to prematurely terminate translation and -1 frame shift. At the pH 5.6 conditions of our structure determination, A 38 is protonated and positively charged in ASL Lys3 UUU -t 6 A 37 and the unmodified ASL Lys3 UUU . The ionized carboxylic acid moiety of t 6 A 37 possibly neutralizes the positive charge of A + 38 . The protonated A + 38 can base 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 unmodified ASL Lys3 UUU would bind as well as ASL Lys3 UUU -t 6 A 37 . t 6 A 37 and other position 37 modifications produce the open, structured loop required for ribosomal binding.Lysine tRNAs with UUU anticodons have a conventional role in ribosome-mediated protein synthesis. In addition, tRNA Lys UUU species facilitate -1 frameshifts for correct translation of the E. coli DNA polymerase γ subunit (1) and retroviral polymerases (2). Also, tRNA Lys UUU often misreads asparagine codons (3, 4) and peptidyl-tRNA Lys prematurely terminates translation more often than other tRNAs (5). In addition, reverse transcription of the HIV-1 genomic RNA is primed by the human tRNA Lys3 UUU . Formation of the viral replication initiation complex is enhanced in vitro by the pr...

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