Understanding the Effect of Post Transcriptional Modifications in the Anticodon Stem Loop of E.coli tRNA Arginine

Post-transcriptional modifications in the anticodon stem loop (ASL) of tRNAs are known to affect their conformation, thermal stability, ribosomal binding and decoding specificity. Escherichia coli uses only two tRNAArg isoacceptors to decode three of the six arginine codons (CGU, CGC and CGA). The ASLs of both the isoacceptors (tRNAArg1,2) contain naturally occurring modifications at position 34 (Inosine, I34) and 37 (2-methyladenosine, m2A37). In addition, tRNA Arg1 is post-transcriptionally modified to contain a 2-thiocytidine (s2C32) modification. To investigate the functional roles of these modifications, six ASL constructs, differing in their array of modifications, were analyzed using binding studies, as well as structural and computational methods. Ribosome filter binding assays showed that while I34, as expected, facilitates wobble codon binding, both s2C32 and m2A37 modulates that effect by negating binding to the rare CGA codon. Similar results were observed when a non-naturally occurring s2C32 was introduced for C32 in an unrelated S. cerevisiae tRNA ASLIle construct also containing I34 capable of wobble pairing. However, the solution structures show minor variation in the anticodon stem loop. The mechanism by which the modification affects codon binding was further investigated by molecular dynamics simulations. Using free-energy perturbation calculations we found that the s2C32 modification destabilizes the anticodon stem loop in solution in agreement with the experiments. Our simulations show that there is a free energy penalty for introducing s2C32 in ribosome bound tRNA explaining the reduction in binding observed experimentally. The simulation results are consistent with the experiments and provide a platform for gaining insights into the molecular explanation for how post-transcriptional modifications modulate tRNA function.