Post‐transcriptional Modifications Alter Anticodon Loop Dynamics and Codon Recognition in E. coli tRNAArg1,2

Abstract

In Escherichia coli, three of six arginine codons (CGU, CGC and CGA) are decoded by only two tRNAArg isoacceptors. The anticodon‐stem loop (ASL) domains of tRNAArg1 and tRNAArg2 differ only at position 32: tRNAArg1 is post‐transcriptionally modified to contain a 2‐thiocytidine (s2C32), while the same cytidine in tRNAArg2 is unmodified. Both isoacceptors also contain naturally‐occurring modifications at positions 34 (inosine, I34) and 37 (2‐methyladenosine, m2A37). To investigate the functional roles of these modifications, six ASL constructs, differing in their array of modifications, were analyzed using binding studies and structural and computational methods. Ribosome filter binding assays showed that while I34, as expected, facilitates wobble codon binding, both s2C32 and m2A37 modulated that effect by negating binding to the rare CGA codon. When a non‐naturally occurring s2C32 was introduced for C32 in an unrelated S. cerevisiae tRNA ASLIle construct also containing C32 and I34 and capable of wobble pairing, the same functional restriction of CGA binding was observed. The NMR and x‐ray crystallographic structures of the ASLs failed to offer an explanation for this effect and showed only minor anticodon loop perturbation upon inclusion of s2C32. The mechanism by which the modification affects codon binding was then investigated by molecular dynamics simulations. Using free‐‐energy perturbation calculations, s2C32 was shown to destabilize the anticodon stem loop in solution; a free energy penalty was demonstrated to exist for the introduction of s2C32 in ribosome‐bound tRNA, explaining the reduction in binding observed experimentally. Simulation and experimental results are consistent and provide a platform for gaining insights into the modulation of tRNA function by post‐transcriptional modifications.