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Title: Human tRNA Lys3 UUU Is Pre-Structured by Natural Modifications for Cognate and Wobble Codon Binding through Keto-Enol Tautomerism

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 (mcm 5s 2U 34), 2-methylthio-N 6-threonylcarbamoyladenosine (ms 2t 6A 37), and pseudouridine (Ψ 39) 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-mcm 5s 2U 34;ms 2t 6A 3739). Ribosome binding assays in vitro revealed that the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 3739 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 Ψ 39 enhanced the thermodynamic stability of the ASL through base stacking while ms 2t 6A 37 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, orderedmore » loop for codon binding. The crystal structures of the hASL Lys3 UUU-mcm 5s 2U 34;ms 2t 6A 3739 bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm 5s 2U 34 and ms 2t 6A 37 participate in the stability of the anticodon–codon interaction. Importantly, the mcm 5s 2U 34·G 3 wobble base pair is in the Watson–Crick geometry, requiring unusual hydrogen bonding to G in which mcm 5s 2U 34 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.« less
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Publication Date:
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Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology; Journal Volume: 416; Journal Issue: 4
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org:
Country of Publication:
United States