Genes VII

Isolation of mutant tRNAs has been one of the most potent tools for analyzing the ability of a tRNA to respond to its codon(s) in mRNA, and for determining the effects that different parts of the tRNA molecule have on codon-anticodon recognition.

Mutant tRNAs are isolated by virtue of their ability to overcome the effects of mutations in genes coding for proteins. We have already described the terminology in which a mutation that is able to overcome the effects of another is called a suppressor (see 1 Genes are DNA).

In tRNA suppressor systems, the primary mutation changes a codon in an mRNA so that the protein product is no longer functional. The secondary, suppressor mutation changes the anticodon of a tRNA, so that it recognizes the mutant codon instead of (or as well as) its original target codon. The amino acid that is now inserted restores protein function. The suppressors are named as nonsense or missense, depending on the nature of the original mutation.

In a wild-type cell, a nonsense mutation is recognized only by a release factor, terminating protein synthesis. The suppressor mutation creates an aminoacyl-tRNA that can recognize the termination codon; by inserting an amino acid, it allows protein synthesis to continue beyond the site of nonsense mutation.

This new capacity of the translation system allows a full-length protein to be synthesized, as illustrated in Figure 7.18. If the amino acid inserted by suppression is different from the amino acid that was originally present at this site in the wild-type protein, the activity of the protein may be altered.

Nonsense suppressors fall into three classes, one for each type of termination codon. Figure 7.19 describes the properties of some of the best characterized suppressors.

The easiest to characterize have been amber suppressors. In E. coli, at least 6 tRNAs have been mutated to recognize UAG codons. All of the amber suppressor tRNAs have the anticodon CUA^←, in each case derived from wild type by a single base change. The site of mutation can be any one of the three bases of the anticodon, as seen from supD, supE, and supF. Each suppressor tRNA recognizes only the UAG codon, instead of its former codon(s). The amino acids inserted are serine, glutamine, or tyrosine, the same as those carried by the corresponding wild-type tRNAs.

Ochre suppressors also arise by mutations in the anticodon. The best known are supC and supG, which insert tyrosine or lysine in response to both ochre (UAA) and amber (UAG) codons. This conforms with the prediction of the wobble hypothesis that UAA cannot be recognized alone.

A UGA suppressor has an unexpected property. It is derived from tRNA^Trp, but its only mutation is the substitution of A in place of G at position 24. This change replaces a G PU pair in the D stem with an A PU pair, increasing the stability of the helix. The sequence of the anticodon remains the same as the wild type, CCA^← . So the mutation in the D stem must in some way alter the conformation of the anticodon loop, allowing CCA^← to pair with UGA in an unusual wobble pairing of C with A. The suppressor tRNA continues to recognize its usual codon, UGG.

A related response is seen with a eukaryotic tRNA. Bovine liver contains a tRNA^Ser with the anticodon ^mCCA^←. The wobble rules predict that this tRNA should respond to the tryptophan codon UGG; but in fact it responds to the termination codon UGA. So it is possible that UGA is suppressed naturally in this situation.

The general importance of these observations lies in the demonstration that codon-anticodon recognition of either wild-type or mutant tRNA cannot be predicted entirely from the relevant triplet sequences, but is influenced by other features of the molecule.

Missense mutations change a codon representing one amino acid into a codon representing another amino acid, one that cannot function in the protein in place of the original residue. (Formally, any substitution of amino acids constitutes a missense mutation, but in practice it is detected only if it changes the activity of the protein.) The mutation can be suppressed by the insertion either of the original amino acid or of some other amino acid that is acceptable to the protein.

Figure 7.20 demonstrates that missense suppression can be accomplished in the same way as nonsense suppression, by mutating the anticodon of a tRNA carrying an acceptable amino acid so that it responds to the mutant codon. So missense suppression involves a change in the meaning of the codon from one amino acid to another.

There is an interesting difference between the usual recognition of a codon by its proper aminoacyl-tRNA and the situation in which mutation allows a suppressor tRNA to recognize a new codon. In the wild-type cell, only one meaning can be attributed to a given codon, which represents either a particular amino acid or a signal for termination. But in a cell carrying a suppressor mutation, the mutant codon has the alternatives of being recognized by the suppressor tRNA or of being read with its usual meaning.

A nonsense suppressor tRNA must compete with the release factors that recognize the termination codon(s). A missense suppressor tRNA must compete with the tRNAs that respond properly to its new codon. The extent of competition influences the efficiency of suppression; so the effectiveness of a particular suppressor depends not only on the affinity between its anticodon and the target codon, but also on its concentration in the cell, and on the parameters governing the competing termination or insertion reactions.

The efficiency with which any particular codon is read is influenced by its location. So the extent of nonsense suppression by a given tRNA can vary quite widely, depending on the context of the codon. We do not understand the effect that neighboring bases in mRNA have on codon-anticodon recognition, but the context can change the frequency with which a codon is recognized by a particular tRNA by more than an order of magnitude. The base on the 3′ side of a codon appears to have a particularly strong effect.

A nonsense suppressor is isolated by its ability to respond to a mutant nonsense codon. But the same triplet sequence constitutes one of the normal termination signals of the cell! The mutant tRNA that suppresses the nonsense mutation must in principle be able to suppress natural termination at the end of any gene that uses this codon. Figure 7.21 shows that this readthrough results in the synthesis of a longer protein, with additional C-terminal material. The extended protein will end at the next termination triplet sequence found in the phase of the reading frame. Any extensive suppression of termination is likely to be deleterious to the cell by producing extended proteins whose functions are thereby altered.

Amber suppressors tend to be relatively efficient, usually in the range of 10 V50%, depending on the system. This efficiency is possible because amber codons are used relatively infrequently to terminate protein synthesis in E. coli.

Ochre suppressors are difficult to isolate. They are always much less efficient, usually with activities below 10%. All ochre suppressors grow rather poorly, which indicates that suppression of both UAA and UAG is damaging to E. coli, probably because the ochre codon is used most frequently as a natural termination signal.

UGA is the least efficient of the termination codons in its natural function; it is misread by Trp-tRNA as frequently as 1 V3% in wild-type situations. In spite of this deficiency, however, it is used more commonly than the amber triplet to terminate bacterial genes.

One gene’s missense suppressor is likely to be another gene’s mutator. A suppressor corrects a mutation by substituting one amino acid for another at the mutant site. But in other locations, the same substitution will replace the wild-type amino acid with a new amino acid. The change may inhibit normal protein function.

This poses a dilemma for the cell: it must suppress what is a mutant codon at one location, while failing to change too extensively its normal meaning at other locations. The absence of any strong missense suppressors is therefore explained by the damaging effects that would be caused by a general and efficient substitution of amino acids.

A mutation that creates a suppressor tRNA can have two consequences. First, it allows the tRNA to recognize a new codon. Second, sometimes it prevents the tRNA from recognizing the codons to which it previously responded. It is significant that all the high-efficiency amber suppressors are derived by mutation of one copy of a redundant tRNA set. In these cases, the cell has several tRNAs able to respond to the codon originally recognized by the wild-type tRNA. So the mutation does not abolish recognition of the old codons, which continue to be served adequately by the tRNAs of the set. (In the unusual situation in which there is only a single tRNA that responds to a particular codon, any mutation that prevents the response is lethal (for review see 31, 36, 37, 38).