October 16, 2012

Suppressors may compete with wild-type reading of the code

  • The context of a codon in mRNA refers to the fact that neighboring sequences may change the efficiency with which a codon is recognized by its aminoacyl-tRNA or is used to terminate protein synthesis.
  • Readthrough at transcription or translation occurs when RNA polymerase or the ribosome, respectively, ignores a termination signal because of a mutation of the template or the behavior of an accessory factor.
  • Suppressor tRNAs compete with wild-type tRNAs that have the same anticodon to read the corresponding codon(s).
  • Efficient suppression is deleterious because it results in readthrough past normal termination codons.
  • The UGA codon is leaky and is misread by Trp-tRNA at 1-3% frequency. 

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 7.26 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 10-50%, 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 1-3% 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 Murgola, 1985; Eggertsson and Soll, 1988; Normanly and Abelson, 1989; Atkins, 1991).
Suppression is most often considered in the context of a mutation that changes the reading of a codon. However, there are some situations in which a stop codon is read as an amino acid at a low frequency in the wild-type situation. The first example to be discovered was the coat protein gene of the RNA phage Qβ. The formation of infective Qβ particles requires that the stop codon at the end of this gene is suppressed at a low frequency to generate a small proportion of coat proteins with a C-terminal extension. In effect, this stop codon is leaky. The reason is that Trp-tRNA recognizes the codon at a low frequency (Hirsh, 1971; Weiner and Weber, 1973).
Readthrough past stop codons occurs also in eukaryotes, where it is employed most often by RNA viruses. This may involve the suppression of UAG/UAA by Tyr-tRNA, Gln-tRNA, or Leu-tRNA, or the suppression of UGA by Trp-tRNA or Arg-tRNA. The extent of partial suppression is dictated by the context surrounding the codon (for review see Beier and Grimm, 2001).