"U-turns" represent an important class of structural motifs in the RNA worl
d, wherein a uridine is involved in an abrupt change in the direction of th
e polynucleotide backbone. In the crystal structure of yeast tRNA(Phe), the
invariant uridine at position 33 (U-33), adjacent to the anticodon, stabil
izes 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 ea
ch noncanonical interaction was determined by assaying the ribosomal bindin
g affinities of tRNA(Phe) anticodon stem and loop domains (ASLs) with subst
itutions 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 n
M and 2,190 +/- 300 nM, respectively). Surprisingly, the ASL with N-3-methy
luridine-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 n
onstacking, nonnative, and constrained conformations, dihydrouridine (C2'-e
ndo), 6-methyluridine (syn) and 2'O-methyluridine (C3'-endo) had almost und
etectable binding. The inability of ASLs with 6-methyluridine-33 and 2'O-me
thyluridine-33 to bind ribosomes was not attributable to any thermal instab
ility 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 bind
ing. Rather, the results suggest that the overall uridine conformation, inc
luding 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 contr
ibuting factors to a functional U-turn.