KEY TERMS:
- Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain.
- Kinetic proofreading describes a proofreading mechanism that depends on incorrect events proceeding more slowly than correct events, so that incorrect events are reversed before a subunit is added to a polymeric chain.
- Chemical proofreading describes a proofreading mechanism in which the correction event occurs after addition of an incorrect subunit to a polymeric chain, by reversing the addition reaction.
- Specificity of recognition of both amino acid and tRNA is controlled by aminoacyl-tRNA synthetases by proofreading reactions that reverse the catalytic reaction if the wrong component has been incorporated.
Aminoacyl-tRNA synthetases have a difficult job. Each
synthetase must distinguish 1 out of 20 amino acids, and and must differentiate
cognate tRNAs (typically 1-3) from the total set (perhaps 100 in all).
Many amino acids are closely related to one another, and all
amino acids are related to the metabolic intermediates in their particular
synthetic pathway. It is especially difficult to distinguish between two amino
acids that differ only in the length of the carbon backbone (that is, by one
CH2 group). Intrinsic discrimination based on relative energies of
binding two such amino acids would be only ~1/5. The synthetase enzymes improve
this ratio ~1000 fold.
Intrinsic discrimination between tRNAs is better, because
the tRNA offers a larger surface with which to make more contacts, but it is
still true that all tRNAs conform to the same general structure, and there may
be a quite limited set of features that distinguish the cognate tRNAs from the
noncognate tRNAs.
We can imagine two general ways in which the enzyme might
select its substrate:
- The cycle of admittance, scrutiny, rejection/acceptance could represent a single binding step that precedes all other stages of whatever reaction is involved. This is tantamount to saying that the affinity of the binding site is sufficient to control the entry of substrate. In the case of synthetases, this would mean that only the correct amino acids and cognate tRNAs could form a stable attachment at the site.
- Alternatively, the reaction proceeds through some of its stages, after which a decision is reached on whether the correct species is present. If it is not present, the reaction is reversed, or a bypass route is taken, and the wrong member is expelled. This sort of postbinding scrutiny is generally described as proofreading. In the example of synthetases, it would require that the charging reaction proceeds through certain stages even if the wrong tRNA or amino acid is present.
Synthetases use proofreading mechanisms to control the
recognition of both types of substrates. They improve significantly on the
intrinsic differences among amino acids or among tRNAs, but, consistent with the
intrinsic differences in each group, make more mistakes in selecting amino acids
(error rates are 10–4 - 10–5) than in selecting tRNAs (error rates are
~10-6) (see Figure 6.8).
Transfer RNA binds to synthetase by the two stage reaction
depicted in Figure 7.18. Cognate tRNAs have a greater
intrinsic affinity for the binding site, so they are bound more rapidly and
dissociate more slowly. Following binding, the enzyme scrutinizes the tRNA that
has been bound. If the correct tRNA is present, binding is stabilized by a
conformational change in the enzyme. This allows aminoacylation to occur
rapidly. If the wrong tRNA is present, the conformational change does not occur.
As a result, the reaction proceeds much more slowly; this increases the chance
that the tRNA will dissociate from the enzyme before it is charged. This type of
control is called kinetic proofreading (Hopfield, 1974).
Specificity for amino acids varies among the synthetases.
Some are highly specific for initially binding a single amino acid, but others
can also activate amino acids closely related to the proper substrate. Although
the analog amino acid can sometimes be converted to the adenylate form, in none
of these cases is an incorrectly activated amino acid actually used to form a
stable aminoacyl-tRNA.
The presence of the cognate tRNA usually is needed to
trigger proofreading, even if the reaction occurs at the stage before formation
of aminoacyl-adenylate. (An exception is provided by Met-tRNA synthetase, which
can reject noncognate aminoacyl-adenylate complexes even in the absence of
tRNA.)
There are two stages at which proofreading of an incorrect
aminoacyl-adenylate may occur during formation of aminoacyl-tRNA. Figure 7.19 shows that both use chemical proofreading, in which the catalytic reaction is
reversed. The extent to which one pathway or the other predominates varies with
the individual synthetase:
- The noncognate aminoacyl-adenylate may be hydrolyzed when the cognate tRNA binds. This mechanism is used predominantly by several synthetases, including those for methionine, isoleucine, and valine. (Usually, the reaction cannot be seen in vivo, but it can be followed for Met-tRNA synthetase when the incorrectly activated amino acid is homocysteine, which lacks the methyl group of methionine). Proofreading releases the amino acid in an altered form, as homocysteine thiolactone. In fact, homocysteine thiolactone is produced in E. coli as a by-product of the charging reaction of Met-tRNA synthetase. This shows that continuous proofreading is part of the process of charging a tRNA with its amino acid (Jakubowski, 1990).
- Some synthetases use chemical proofreading at a later stage. The wrong amino acid is actually transferred to tRNA, is then recognized as incorrect by its structure in the tRNA binding site, and so is hydrolyzed and released. The process requires a continual cycle of linkage and hydrolysis until the correct amino acid is transferred to the tRNA.
A classic example in which discrimination between amino
acids depends on the presence of tRNA is provided by the Ile-tRNA synthetase of
E. coli. The enzyme can charge valine with AMP, but hydrolyzes the
valyl-adenylate when tRNAIle is added. The overall error rate depends
on the specificities of the individual steps, as summarized in Figure 7.20. The overall error rate of 1.5 × 10–5 is less
than the measured rate at which valine is substituted for isoleucine (in rabbit
globin), which is 2-5 × 10–4. So mischarging probably provides only a small
fraction of the errors that actually occur in protein synthesis.
Ile-tRNA synthetase uses size as a basis for discrimination
among amino acids. Figure 7.21 shows that it has two
active sites: the synthetic (or activation) site and the editing (or hydrolytic)
site. The crystal structure of the enzyme shows that the synthetic site is too
small to allow leucine (a close analog of isoleucine) to enter. All amino acids
large than isoleucine are excluded from activation because they cannot enter the
synthetic site. An amino acid that can enter the synthetic site is placed on
tRNA. Then the enzyme tries to transfer it to the editing site. Isoleucine is
safe from editing because it is too large to enter the editing site. However,
valine can enter this site, and as a result an incorrect Val-tRNAIle
is hydrolyzed. Essentially the enzyme provides a double molecular sieve, in
which size of the amino acid is used to discriminate between closely related
species (Nureki et al., 1998; for review see Jakubowski and Goldman, 1992).
One interesting feature of Ile-tRNA synthetase is that the
synthetic and editing sites are a considerable distance apart, ~34Å. A crystal structure of the enzyme complexed with an
edited analog of isoleucine shows that the amino acid is transported from the
synthetic site to the editing site (Silvian, Wang, and Steitz, 1999). Figure 7.22 shows that this involves a change in the
conformation of the tRNA. The amino acid acceptor stem of tRNAIle can
exist in alternative conformations. It adopts an unusual hairpin in order to be
aminoacylated by an amino acid in the synthetic site. Then it returns to the
more common helical structure in order to move the amino acid to the editing
site. The translocation between sites is the rate-limiting step in proofreading
(Nomanbhoy, Hendrickson, and Schimmel, 1999). Ile-tRNA
synthetase is a class I synthetase, but the double sieve mechanism is used also
by class II synthetases (Dock-Bregeon et al., 2000).