Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in organisms are significantly lower.

The high level of accuracy is guaranteed by two additional proofreading steps involving two principles from enzyme-substrate interactions.

Within the ribosome, the peptidyl transferase center (PTC) catalyzes the covalent bond formation between amino acids to form a polypeptide chain. Like any other enzyme, the PTC also has an active site that discriminates between substrates based on their molecular structure. Residues from the 16S rRNA of the small ribosomal subunit form hydrogen bonds with the base and backbone atoms of the codon-anticodon duplex. Only the correct tRNA can induce conformational changes in the PTC, which carries out the catalysis.

The second step, called kinetic proofreading, occurs after the irreversible dissociation of EF-Tu·GDP from the ribosome. GTP hydrolysis marks the start of a short time delay during which the aminoacyl-tRNA moves into the active site of PTC for catalysis. During this delay, any incorrect codon-anticodon pairs that slipped through the induced-fit step are more likely to dissociate than correct pairs. The reason for this is that the wrong tRNA makes weaker base pairs with the codon, and the time delay is longer for incorrect than correct matches.