Following delivery of tRNA to the aminoacyl-tRNA binding site (A site) of the ribosome, a proofreading step ensures that the selected tRNA matches the mRNA codon of the A site. Previous studies suggested that the universally conserved nucleotides G530, A1492 and A1493 of 16S ribosomal RNA (rRNA) promote a conformational change (domain closure) on the 30S ribosomal subunit only when they detect the formation of Watson–Crick base pairs at the first two positions of the codon–anticodon helix. Here, Demeshkina et al. challenge this view showing that 30S domain closure also occurs in the presence of nucleotide mismatches at the codon–anticodon helix. Moreover, G530, A1492 and A1493 nucleotides of the 16S rRNA interact in a similar manner with mismatched base-pairs as with Watson–Crick base pairs. Thus, it seems that it is the formation of a tight decoding centre following 30S domain closure that destabilizesthe incorrect base pairs, leading to the release of near-cognate tRNA.