Solution structure of ψ(32)-modified anticodon stem–loop of Escherichia coli tRNA(Phe)

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ(32), ψ(39), and A(37) C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ(32) on the anticodon stem-loop from E.coli tRNA(Phe). The ψ(32) modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ(32)-A(38) and U(33)–A(37) and the base of ψ(32) stacks between U(33) and A(31). The glycosidic bond of ψ(32) is in the anti configuration and is paired with A(38) in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ(32) modification increases the melting temperature of the stem by ∼3.5°C, although the ψ(32) and U(33) imino resonances are exchange broadened. The results suggest that ψ(32) functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.