Some uses of nonsense suppressors
Nonsense suppressors are widely used genetic tools. Two examples of practical uses of nonsense suppressors are described below.
- Nonsense mutations are examples of conditional mutations -- in a strain lacking a nonsense suppressor (supo), the mutation causes premature termination of protein synthesis, but in a strain with a nonsense suppressor (sup), functional protein can be made. This trick is used in certain cloning vectors. For example, if a phage vector contains nonsense mutations it will only be able to grow in a bacterial host with a nonsense suppressor, limiting potential "escapees" from infecting other bacteria and spreading outside the lab.
- Suppression of nonsense mutations is a simple approach to study the effect of different amino acid substitutions at a specific position in a protein. For example, if a phage mutant with an amber mutation at position #50 of an essential gene infects a supo host no functional protein will be made so the phage will not reproduce; if the phage infects a supD host a serine will be inserted at position #50 of the protein; if the phage infects a supE host a glutamine will be inserted at position #50 of the protein; if the phage infects a supF host a tyrosine will be inserted at position #50 of the protein; and so on. If the amino acid inserted at this position results in a misfolded or otherwise nonfunctional protein, the phage will not reproduce on the suppressor containing host. However, if the amino acid inserted at this site yields a functional protein, the phage will reproduce. By using a collection of natural and synthetic suppressor mutants, it is possible to test the ability of many different amino acids to suppress a specific amber mutant. The results provide useful information about the structure and function of the protein.
Promega sells a nice kit of nonsense suppressors for this type of analysis. A description of it can be found on the
Promega Web site.
- Nonsense suppressors can also be used to incorporate unnatural amino acids into proteins. For example, alpha-hydroxy acids with a wide variety of substituents can be synthesized and attached to a nonsense suppressor tRNA, allowing substitution of the unnatural amino acid for any chosen amino acid residue in the protein. The unnatural amino acids can provide a probe for the role of that position in the structure and function of the protein, etc.
- Dougherty, D. 2000. Unnatural amino acids as probes of protein structure and function. Curr Opin Chem Biol. 4: 645-652. (A nice review describing how nonsense suppressors can be used to incorporate unnatural amino acids into proteins, facilitating novel approaches to study protein structure and function.)
- Kleina LG, Masson JM, Normanly J, Abelson J, Miller JH. 1990. Construction of Escherichia coli amber suppressor tRNA genes. II. Synthesis of additional tRNA genes and improvement of suppressor efficiency.J Mol Biol. 213: 705-717.
- Normanly J, Kleina LG, Masson JM, Abelson J, Miller JH. 1990. Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity. J Mol Biol. 213: 719-726.
- Miller JH, Kleina LG, Masson JM, Normanly J, Abelson J. 1989. Protein engineering with synthetic Escherichia coli amber suppressor genes. Genome. 31: 905-908.
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