Human tRNA^Lys3_UUU Is Pre-Structured by Natural Modifications for Cognate and Wobble Codon Binding through Keto-Enol Tautomerism

Journal Article · Fri Sep 27 00:00:00 EDT 2013 · Journal of Molecular Biology

DOI:https://doi.org/10.1016/j.jmb.2011.12.048· OSTI ID:1094869

Vendeix, Franck A.P.; Cantara, William A.; Leszczy,; ska, Gra; yna,; Gustilo, Estella M.; Sproat, Brian; Malkiewicz, Andrzej; Agris, Paul F. ^[1]

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1. Cornell

Human tRNA^Lys3_UUU (htRNA^Lys3_UUU) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄), 2-methylthio-N⁶-threonylcarbamoyladenosine (ms²t⁶A₃₇), and pseudouridine (Ψ₃₉) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉). Ribosome binding assays in vitro revealed that the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ bound AAA and AAG codons, whereas binding of the unmodified ASL^Lys3_UUU was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that Ψ₃₉ enhanced the thermodynamic stability of the ASL through base stacking while ms²t⁶A₃₇ restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm⁵s²U₃₄ and ms²t⁶A₃₇ participate in the stability of the anticodon–codon interaction. Importantly, the mcm⁵s²U₃₄·G₃ wobble base pair is in the Watson–Crick geometry, requiring unusual hydrogen bonding to G in which mcm⁵s²U₃₄ must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: NSFOTHERNIH
• OSTI ID: 1094869
• Journal Information: Journal of Molecular Biology, Vol. 416, Issue 4; ISSN 0022-2836
• Publisher: Elsevier
• Country of Publication: United States
• Language: ENGLISH