“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 tRNAPhe, 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 tRNAPhe anticodon stem and loop domains (ASLs) with substitutions at U33. An unsubstituted ASL bound 30S ribosomal subunits with an affinity (Kd = 140 ± 50 nM) comparable to that of native yeast tRNAPhe (Kd = 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 (Kd = 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.