Structural Effects of Hypermodified Nucleosides in the Escherichia coli and Human tRNA^Lys Anticodon Loop:  The Effect of Nucleosides s²U, mcm⁵U, mcm⁵s²U, mnm⁵s²U, t⁶A, and ms²t⁶A

Abstract

Previous nuclear magnetic resonance (NMR) studies of unmodified and pseudouridine39-modified tRNA(Lys) anticodon stem loops (ASLs) show that significant structural rearrangements must occur to attain a canonical anticodon loop conformation. The Escherichia coli tRNA(Lys) modifications mnm(5)s(2)U34 and t(6)A37 have indeed been shown to remodel the anticodon loop, although significant dynamic flexibility remains within the weakly stacked U35 and U36 anticodon residues. The present study examines the individual effects of mnm(5)s(2)U34, s(2)U34, t(6)A37, and Mg(2+) on tRNA(Lys) ASLs to decipher how the E. coli modifications accomplish the noncanonical to canonical structural transition. We also investigated the effects of the corresponding human tRNA(Lys,3) versions of the E. coli modifications, using NMR to analyze tRNA ASLs containing the nucleosides mcm(5)U34, mcm(5)s(2)U34, and ms(2)t(6)A37. The human wobble modification has a less dramatic loop remodeling effect, presumably because of the absence of a positive charge on the mcm(5) side chain. Nonspecific magnesium effects appear to play an important role in promoting anticodon stacking. Paradoxically, both t(6)A37 and ms(2)t(6)A37 actually decrease anticodon stacking compared to A37 by promoting U36 bulging. Rather than stack with U36, the t(6)A37 nucleotide in the free tRNAs is prepositioned to form a cross-strand stack with the first codon nucleotide as seen in the recent crystal structures of tRNA(Lys) ASLs bound to the 30S ribosomal subunit. Wobble modifications, t(6)A37, and magnesium each make unique contributions toward promoting canonical tRNA structure in the fundamentally dynamic tRNA(Lys)(UUU) anticodon.

The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

“…Our data showing that overexpression of unmodified tRNA rescues the deletion phenotype and the fact that a decreased level of a synthetic KQ-TAP protein is observed when Ctu1 function is abolished both point to a critical role in the improvement of cognate codon recognition. This requirement of the modification for efficiency might be related to the fact that the corresponding thiolated tRNAs (tRNA LYS UUU , tRNA GLU UUC , and tRNA GLN UUG ) all harbor two uridines in positions 34 and 35 in the anticodon, creating an instable codon-anticodon interaction due to the low stacking potential of uridine (25,(34)(35)(36).…”

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).…”

Nucleotide modifications and tRNA anticodon–mRNA codon interactions on the ribosome

“…The modifications can be situated in all domains of the tRNA and are conserved in many organisms, but the wobble position 34 and the purine 37 on the 39 side of the anticodon stand out as being almost universally modified. The modifications in these places vary greatly in size, ranging from simple methyl groups, like the m 6 A37, to complex structures, like the ms 2 t 6 A37 (Durant et al 2005) but are all, with little doubt, involved in codon recognition (Nishimura and Watanabe 2006). The main effects of the modifications in these two positions are believed to be to open up the anticodon loop by negating intra-loop hydrogen bonds (Olejniczak and Uhlenbeck 2006) and to constrain the dynamics of the loop by increasing the stacking of its bases (Agris 2008 U and its derivatives stand out for FIGURE 1.…”