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 tRNA^Arg1 and tRNA^Arg2 differ only at position 32: tRNA^Arg1 is post-transcriptionally modified to contain a 2-thiocytidine (s²C₃₂), while the same cytidine in tRNA^Arg2 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 ASL^Leu construct also containing C₃₂ and I₃₄ and capable of wobble pairing, the same functional restriction of CGA binding was observed. The structures of the ASLs, when probed by either by NMR spectroscopy or x-ray crystallography, showed only a minor anticodon loop perturbation upon inclusion of s²C₃₂. The mechanism by which the modification affects 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 provide an explanation for 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.

This research has been supported by National Science Foundation award MCB1101859.