Solution structure of ψ32-modified anticodon stem–loop of Escherichia coli tRNAPhe

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 Mg2+ and dimethylallyl modification of A37 N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, psi32, psi39, and A37 C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of psi32 on the anticodon stem-loop from E.coli tRNA(Phe). The psi32 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 psi32-A38 and U33-A37 and the base of psi32 stacks between U33 and A31. The glycosidic bond of psi32 is in the anti configuration and is paired with A38 in a Watson-Crick geometry, unlike residue 32 in most crystal structures of tRNA. The psi32 modification increases the melting temperature of the stem by approximately 3.5 degrees C, although the psi32 and U33 imino resonances are exchange broadened. The results suggest that psi32 functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.