Solution Conformations of Unmodified and A 37N 6-dimethylallyl Modified Anticodon Stem-loops of Escherichia coli tRNA Phe

Abstract

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Δ 2-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A 37 in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA Phe, we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA Phe. The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA Phe anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon–anticodon helix.