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

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 (U-33), adjacent to the anticodon, stabilizes the exemplar U-turn with three non-Watson-Crick interactions: hydrogen bonding of the 2'-OH to N-7 of A(35) and the N-3-H to A(36)-phosphate, and stacking between C-32 and A(35)-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 U-33. 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 N-3-H) were significantly reduced (2,930 +/- 140 nM and 2,190 +/- 300 nM, respectively). Surprisingly, the ASL with N-3-methyluridine-33 (no N-3-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 N-3-H and 2'OH groups of U-33 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 C-32 and A(35)-phosphate, are the contributing factors to a functional U-turn.