Human tRNALys3UUU Is Pre-Structured by Natural Modifications for Cognate and Wobble Codon Binding through Keto–Enol Tautomerism

Human tRNALys3UUU (htRNALys3UUU) 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 tRNAʹs 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 tRNAʹs anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASLLys3UUU-mcm5s2U34;ms2t6A37;Ψ39). Ribosome binding assays in vitro revealed that the hASLLys3UUU-mcm5s2U34;ms2t6A37;Ψ39 bound AAA and AAG codons, whereas binding of the unmodified ASLLys3UUU 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 hASLLys3UUU-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.