A Structural Basis for Restricted Codon Recognition Mediated by 2-thiocytidine in tRNA Containing a Wobble Position Inosine

Three of six arginine codons (CGU, CGC, and CGA) are decoded by two Escherichia coli tRNA^Arg isoacceptors. The anticodon stem and loop (ASL) domains of tRNA^Arg1 and tRNA^Arg2 both contain inosine and 2-methyladenosine modifications at positions 34 (I₃₄) and 37 (m²A₃₇). tRNA^Arg1 is also modified from cytidine to 2-thiocytidine at position 32 (s²C₃₂). The s²C₃₂ modification is known to negate wobble codon recognition of the rare CGA codon by an unknown mechanism, while still allowing decoding of CGU and CGC. Substitution of s²C₃₂ for C₃₂ in the Saccharomyces cerevisiae tRNA^Ile _IAU anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of s²C at position 32 is a generalizable property. X-ray crystal structures of variously modified ASL^Arg1 _ICG and ASL^Arg2 _ICG constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a C_32-A₃₈ cross-loop interaction but failed to fully explain the means by which s²C₃₂ restricts I₃₄ wobbling. Computational studies revealed that the adoption of a spatially broad inosine-adenosine base pair at the wobble position of the codon cannot be maintained simultaneously with the canonical ASL U-turn motif. C_32-A₃₈ cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.