Modifications Modulate Anticodon Loop Dynamics and Codon Recognition in E. coli tRNAArg1,2

Abstract

In E. 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 posttranscriptionally modified to contain a 2‐thiocytidine (s2C32). Both isoacceptors also contain naturally‐occurring inosine (I34) and 2‐methyladenosine (m2A­­37) modifications. To investigate the functional roles of these modifications, six ASL constructs differing in modification profile were analyzed using binding studies and structural and computational methods. Ribosome filter binding assays showed that while I34 facilitates wobble codon binding, both s2C32 and m2A37 modulate 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 ASLLeu construct also containing I34, the same functional restriction of CGA binding was observed. The NMR and x‐ray crystallographic structures of the ASLs showed only a minor anticodon loop perturbation upon inclusion of s2C32. The mechanism of its effect on codon binding was then investigated by molecular dynamics simulations, which suggested that both s2C32 and m2A37 afford the ASL greater flexibility and conformational accessibility and explained their ability to adopt one structure free in solution and two others when bound to the cognate arginyl‐tRNA synthetase or to codons on the ribosome.