Base Pairing within the ψ_(32),ψ_(39)-Modified Anticodon Arm of Escherichia coli tRNA~(Phe)

摘要

The anticodon arm of tRNA is central to the accuracy and efficiency of protein synthesis. In addition to the three anticodon nucleotides, the extended anticodon hypothesis predicts that the sequence and modification state of nucleotides in the loop-proximal region of the anticodon arm can modulate the decoding function of tRNA. Consistent with this hypothesis is the influence that nucleotides 32 and 38 can have on the accuracy of translation and on the affinity of tRNA for the ribosome. The accuracy conferred by these residues suggests they have a role in fine-tuning the conformational or thermodynamic features of the anticodon arm either in the free tRNA or in the ribosome-bound tRNA. The crystal structures of yeast tRNA~(Phe), tRNA~(Asp), and several tRNA-protein complexes reveal the presence of a conserved motif, the bifurcated hydrogen bond, between nucleotides 32 and 38. The bifurcated hydrogen bond allows for an isosteric arrangement of nucleotides 32 and 38 that is preserved among C_(32)-A_(38), ψ_(32)-A_(38), and ψ_(32)- C_(38) interactions. This arrangement leads to partial unwinding of the helix and may help promote formation of the U-turn motif of the anticodon loop. The unmodified anticodon arm of E. coli tRNA~(Phe) also is highly ordered in solution. However, the anticodon adopts a triloop conformation rather than the characteristic U-turn, and U_(32) and A_(38) form a Watson-Crick base pair instead of the bifurcated hydrogen bond. To determine if the native modifications ψ_(32) and ψ_(39) in the anticodon arm lead to formation of the bifurcated hydrogen-bond motif or the U-turn, we have used heteronuclear NMR spectroscopy to investigate their effects on the local hydrogen-bond geometry between nucleotides 32 and 38 and on the global architecture of the anticodon loop.