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

Abstract

Human tRNALys3UUU (htRNALys3UUU) decodes the lysine codons AAA and AAG during translation, and also plays a crucial role as the primer for HIV-1 reverse transcription. The post-transcriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A37)and pseudouridine (ψ39) in the tRNA'santicodon loop are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, the structure and function of this tRNA's anticodon stem and loop domain were determined with these modifications at positions 34, 37 and 39, respectively (hASLLys3UUU-mcm5s2U37;ms2t6A37;ψ39). Ribosome binding assays in vitrorevealed that the hASLLys3UUU-mcm5s2U34;ms2t6A37;ψ39bound 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 enol form. The results unambiguously demonstrate that modifications pre-structurethe anticodonas a key prerequisite for efficient and accurate recognition of cognate and wobble codons.

Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

“…9 An explanation for the dependence of the mcm 5 s 2 U 34 nucleoside is that it promotes a canonical anticodon loop conformation which stabilize codon-anticodon interaction. 29,30 Additional support that elevated levels of hypomodified tRNA correct phenotypes observed in mutants with defects in wobble uridine modifications came from experiments in Schizosaccharomyces pombe. In S. pombe, elevated levels of hypomodified tRNA Lys mcm 5 s 2 UUU or tRNA Glu mcm 5 s 2 UUC or combinations thereof suppress phenotypes observed in the ctu1D and ctu2D single mutants lacking the s 2 -group, the elp3/sin3D single mutant or the elp3D ctu1D double mutant lacking both modifications ( Table 1).…”

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…Both U36 and A37 are essential for modification and A38 enhances the rate of the modification reaction (5). The t 6 A 37 modification has been shown to have an important role in ribosome-mediated codon binding for several tRNA species (6 -8), mainly due to its ability to enhance the stability of the anticodon-codon base pairing by creating cross-strand basestacking interactions with the first position of the codon, as depicted in structural analyses of tRNA Lys UUU decoding at the ribosome A-site (9,10). This stabilization is necessary to over-come the low enthalpy of binding for U-A base pairs and tRNA Lys UUU in particular with three U-A base pairs (11).…”

Celebrating wobble decoding: Half a century and still much is new

“…In S cerevisiae , loss of either the xm 5 or s 2 modifications of mcm 5 s 2 U 34 increases observation of +1 translational frameshifts, 133 probably caused by a decrease in the affinity of the tRNA for the ribosomal A-site and an impaired translocation. 9,129 The nearly ubiquitous t 6 A 37 modification at position 37 in tRNAs responding to codons beginning with A, ANN, enhances base stacking of U 36 with A1 of the codon, as well as prevents intraloop base pairing within the ASL. 115 Thereby, t 6 A 37 facilitates the U-turn and presentation of the anticodon to the codon on the ribosome.…”

“…Individual anticodon domain modified nucleosides are identity determinants for protein recognition, particularly aminoacylation, 15-17 increase accuracy and efficiency in codon recognition, 18-24 and pre-structure the ASL for translation. 9,25-30 Each of the modified nucleosides contribute distinct chemistries, nucleoside conformations and dynamics, and their contributions to decoding have been studied extensively over decades and most recently reviewed. 20,22,24,28,31-38 However, there is significant evidence that a combination of two or three anticodon domain modifications play a synergistic role in tRNA function where modification of a wobble position U is crucial.…”

“…20,22,24,28,31-38 However, there is significant evidence that a combination of two or three anticodon domain modifications play a synergistic role in tRNA function where modification of a wobble position U is crucial. 9,20,24,39-46 Today, we know that certain modifications of U 34 enable expansion of codon from NNA/G recognition to synonymous codons ending in pyrimidines, N1-N2-Pyr, where N is any of the 4 nucleosides and Pyr is either U or C. 29 Yet, the anticodon domain of some tRNA species lack modification and can be totally devoid of modified nucleosides. Bacteriophage T4 tRNA Gly is an early example of a tRNA lacking modification.…”