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

Previous nuclear magnetic resonance (NMR) studies of unmodified and ψ39-modified tRNALys anticodon stem loops (ASLs) show that significant structural rearrangements must occur to attain a canonical anticodon loop conformation. The Escherichia coli tRNALys modifications mnm5s2U34 and t6A37 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 mnm5s2U34, s2U34, t6A37, and Mg2+ on tRNALys ASLs to decipher how the E. coli modifications accomplish the noncanonical to canonical structural transition. We also investigated the effects of the corresponding human tRNALys,3 versions of the E. coli modifications, using NMR to analyze tRNA ASLs containing the nucleosides mcm5U34, mcm5s2U34, and ms2t6A37. The human wobble modification has a less dramatic loop remodeling effect, presumably because of the absence of a positive charge on the mcm5 side chain. Nonspecific magnesium effects appear to play an important role in promoting anticodon stacking. Paradoxically, both t6A37 and ms2t6A37 actually decrease anticodon stacking compared to A37 by promoting U36 bulging. Rather than stack with U36, the t6A37 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 tRNALys ASLs bound to the 30S ribosomal subunit. Wobble modifications, t6A37, and magnesium each make unique contributions toward promoting canonical tRNA structure in the fundamentally dynamic tRNALysUUU anticodon.