The uridine in “U-turn”: Contributions to tRNA-ribosomal binding

Abstract

“U-turns” represent an important class of structural motifs in the RNA world, wherein a uridine is involved in an abrupt change in the direction of the polynucleotide backbone. In the crystal structure of yeast tRNA^Phe, the invariant uridine at position 33 (U₃₃), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson–Crick interactions: hydrogen bonding of the 2′-OH to N⁷ of A₃₅ and the N³-H to A₃₆-phosphate, and stacking between C₃₂ and A₃₅-phosphate. The functional importance of each noncanonical interaction was determined by assaying the ribosomal binding affinities of tRNA^Phe anticodon stem and loop domains (ASLs) with substitutions at U₃₃. An unsubstituted ASL bound 30S ribosomal subunits with an affinity (K_d = 140 ± 50 nM) comparable to that of native yeast tRNA^Phe (K_d = 100 ± 20 nM). However, the binding affinities of ASLs with dU-33 (no 2′-OH) and C-33 (no N³-H) were significantly reduced (2,930 ± 140 nM and 2,190 ± 300 nM, respectively). Surprisingly, the ASL with N³-methyluridine-33 (no N³-H) bound ribosomes with a high affinity (K_d = 220 ± 20 nM). In contrast, ASLs constructed with position 33 uridine analogs in nonstacking, nonnative, and constrained conformations, dihydrouridine (C2′-endo), 6-methyluridine (syn) and 2′O-methyluridine (C3′-endo) had almost undetectable binding. The inability of ASLs with 6-methyluridine-33 and 2′O-methyluridine-33 to bind ribosomes was not attributable to any thermal instability of the RNAs. These results demonstrate that proton donations by the N³-H and 2′OH groups of U₃₃ are not absolutely required for ribosomal binding. Rather, the results suggest that the overall uridine conformation, including a dynamic (C3′-endo > C2′-endo) sugar pucker, anti conformation, and ability of uracil to stack between C₃₂ and A₃₅-phosphate, are the contributing factors to a functional U-turn.