The first is induced fit. We have seen that, before an amino acid is added to a growing polypeptide chain, the ribosome folds around the codon–anticodon interaction, and only when the match is correct is this folding completed and the reaction allowed to proceed. Thus, the codon–anticodon interaction is thereby checked twice—once by the initial complementary base-pairing and a second time by the folding of the ribosome, which depends on the correctness of the match. This same principle of induced fit is seen in transcription by RNA polymerase; here, an incoming nucleoside triphosphate initially forms a base pair with the template; at this point the enzyme folds around the base pair (thereby assessing its correctness) and, in doing so, creates the active site of the enzyme. The enzyme then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being incorporated into the growing chain.
A second principle used to increase the specificity of complementary base-pairing is called kinetic proofreading. We have seen that after the initial codon‒anticodon pairing and conformational change of the ribosome, GTP is hydrolyzed. This creates an irreversible step and starts the clock on a time delay during which the aminoacyl-tRNA moves into the proper position for catalysis. During this delay, those incorrect codon–anticodon pairs that have somehow slipped through the induced-fit scrutiny have a higher likelihood of dissociating than correct pairs. There are two reasons for this: (1) the interaction of the wrong tRNA with the codon is weaker, and (2) the delay is longer for incorrect than correct matches.
In its most general form, kinetic proofreading refers to a time delay that begins with an irreversible step such as ATP or GTP hydrolysis, during which an incorrect substrate is more likely to dissociate than a correct one. In this case, kinetic proofreading thus increases the specificity of complementary base-pairing above what is possible from simple thermodynamic associations alone. The increase in specificity produced by kinetic proofreading comes at an energetic cost in the form of ATP or GTP hydrolysis. Kinetic proofreading is believed to operate in many biological processes, but its role is understood particularly well for translation.
Accuracy in Translation Requires an Expenditure of Free Energy
Translation by the ribosome is a compromise between the opposing constraints of accuracy and speed. We have seen, for example, that the accuracy of translation (1 mistake per 104 amino acids joined) requires time delays each time a new amino acid is added to a growing polypeptide chain, producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.
We have also seen that attaining the observed accuracy of protein synthesis requires the expenditure of a great deal of free energy; this is expected, since, as discussed in Chapter 2, there is a price to be paid for any increase in order in the cell. In most cells, protein synthesis consumes more energy than any other biosynthetic process. At least four high-energy phosphate bonds are split to make each new peptide bond: two are consumed in charging a tRNA molecule with an amino acid (see Figure 6–54), and two more drive steps in the cycle of reactions occurring on the ribosome during protein synthesis itself (see Figure 6–65). In addition, extra energy is consumed each time that an incorrect amino acid linkage is hydrolyzed by a tRNA synthetase (see Figure 6–57) and each time that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected (see Figure 6–65). To be effective, any proofreading mechanism must also allow an appreciable fraction of correct interactions to be removed; for this reason, proofreading is even more costly in energy than it might at first seem.
The Ribosome Is a Ribozyme