Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome.

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.