Ribosomal ambiguity (ram) mutations promote 30S domain closure and thereby increase miscoding

During protein synthesis, ribosomes select correct aminoacyl-tRNAs by monitoring the nucleotide pairing between the codon on the mRNA and the anticodon of the tRNA in the decoding center. Despite the large pool of near-cognate tRNAs, the ribosome is highly accurate with an error rate of ~10−3 – 10−5. The process of decoding by the ribosome is thought to be governed by a conformational transition in the ribosome from open to closed that occurs upon codon-anticodon pairing within the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to both ribosomal proteins and rRNA in disparate regions, yet precisely how these mutations act has remained unclear. In this study, we solved crystal structures of Thermus thermophilus 70S ribosomes harboring 16S rRNA ram mutations G299A and G347U in the absence of A-site tRNA (A-tRNA) and in the presence of a near-cognate anticodon stem-loop. In the absence of an A-tRNA, each of the mutant ribosomes exhibits a partially closed state. In the 70S-G347U struc...