Abstract:

<p>Three of six arginine codons (CGU, CGC and CGA) are decoded by two Escherichia coli tRNA isoacceptors. The anticodon stem and loop (ASL) domains of tRNA and tRNA both contain inosine and 2-methyladenosine modifications at positions 34 (I) and 37 (mA). tRNA is also modified from cytidine to 2-thiocytidine at position 32 (sC). The sC 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 sC for C in the Saccharomyces cerevisiae tRNA anticodon stem and loop domain (ASL) negates wobble decoding of its synonymous A-ending codon, suggesting that this function of sC at position 32 is a generalizable property. X-ray crystal structures of variously modified ASL and ASL constructs bound to cognate and wobble codons on the ribosome revealed the disruption of a CA cross-loop interaction, but failed to fully explain the means by which sC restricts I 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. CA cross-loop interactions are required for stability of the anticodon/codon interaction in the ribosomal A-site.</p>