Genetic Suppression

 Genetic suppression is the relief of a mutant phenotype by a second mutation. The second mutation gives the appearance of “suppressing” the first mutation. The suppressor mutation identifies a new site affecting the phenotype of interest, and suppression is strong evidence that the two genetic sites functionally interact. Suppressor mutations have been extremely useful for understanding genetic and biochemical phenomena. Biological systems are a tangle of interacting molecules and intersecting pathways. It should come as no surprise that mutations in one molecule are often modulated by secondary mutations in others.

 Suppressor mutations can occur in the same gene as the initial mutation (intragenic suppression), or the two mutations may be in separate genes (intergenic suppression). Intragenic suppression may occur if the amino acid residues specified by the two sites must interact to give the normal function of the gene product. Intragenic suppression may also occur by the relief of a polar effect on gene expression. Intergenic suppression has been extensively used to characterize interactions between gene products. In certain cases, the suppressor mutation may establish a new pathway that supplants the pathway disrupted by the initial mutation. A special kind of suppression occurs when mutants are “rescued” by normal genes expressed from high-copy plasmids. The high concentration may facilitate an interaction between proteins that is weakened by the first mutation, or it may provide an alternative pathway to a normal phenotype. Another special kind of intergenic suppression occurs when mutations occur in transfer RNAs so that they translate stop (nonsense codons) codons as sense codons. These tRNAs (nonsense suppressors) can allow expression of genes that have suffered mutations that create stop codons within coding sequences. See “Nonsense suppression” and “tRNA suppressor” for more complete descriptions of these phenomena.

Suppressors were initially detected during studies of mutations. It was found that certain mutations express altered phenotypes only in certain genetic backgrounds. Apparently, in the non-expressing strains other genes were able to suppress these mutations. The molecular basis of these early observations has remained obscure (1), but they have been instructive for defining the principle of suppression.

In a now classic study, intragenic suppression was used to study the fundamental nature of the genetic code. Francis Crick et al. (2) showed that genes are expressed as triplets that do not overlap one another. These workers first showed that mutations that were caused by the insertion (+) or deletion (–) of nucleotides could be suppressed by a second mutation of the opposite sign, if the two mutations occurred near one another within the gene. It was concluded that genes must be read in a specific “reading frame”. Either an insertion or a deletion would shift the reading frame such that the remainder of message sequence is decoded to specify the wrong polypeptide sequence. These workers further concluded that the second mutation of the opposite sign suppresses the first mutation by restoring the initial reading frame. In between the two mutations, the gene would be translated in a wrong reading frame, but as long as the amino acid sequence of that region was not critical for protein function then the double mutant would have a wild type phenotype. Crick also used suppressor mutations to determine the size of the “codon”. It was found that although one or two insertions invariably gave a mutant phenotype, three insertions or three deletions could give a wild type phenotype. It was concluded that because the insertion or deletion of three bases did not perturb a reading frame, the genetic code is a triplet code.

Intragenic suppression can also be used to show that two amino acid within a protein molecule interact. A mutation that eliminates a critical interaction between two amino acids can sometimes be suppressed by a change in the second amino acid that restores complementarity. The first example of such an analysis is presented by Helinsky and Yanofsky (3), in which an intragenic second-site mutation restored partial function to an Escherichia coli trpA mutant. Such information can complement structural studies by providing confirmation of hypothetical interactions between amino acids at specific positions. Such work is greatly facilitated by site-directed mutagenesis, which allows the creation of specific mutations to test structural predictions.

 Intergenic suppression is extensively used to identify and study interactions between molecules. The principle is that mutations that destroy complementary interactions between proteins may be compensated by secondary mutations that provide suitable alternative interactions. This approach has been used to identify proteins interact within biochemical, gene expression, and signal transduction pathways. This approach is also widely used to study interactions between molecules already known or suspected to interact. Examples include studies of receptor-ligand interactions and of interactions between transcription factors and promoter elements.

Another commonly used method is the identification of cloned genes that can suppress mutations when expressed at high levels. Cells are transformed with expression libraries, and suppressed isolates are selected. Suppression may occur when the overexpressed protein compensates for the defect of the initial mutation. Several molecular mechanisms are possible, and further work is usually required to elucidate the mechanism of suppression in each instance. Possible mechanisms include the restoration of a direct interaction between proteins, increasing the expression of a partially-active mutant protein, and providing a bypass of the cellular or biochemical defect of the primary mutation. A related approach, though not usually thought of as suppression, is the use of the various “two-hybrid” systems to identify interacting proteins. These are actually highly-derived examples of intergenic suppression.

Another special but widely employed kind of intergenic suppression occurs when tRNAs are mutated such that they insert a suitable amino acid at missense, nonsense and frameshift mutations. These “informational” or “translational” suppressors have been extremely useful for studying the decoding mechanisms (4). Currently, certain translational suppressors are in development as tools for protein engineering. Systems for labeling proteins with novel amino acids at specific positions would be extremely useful. Toward this end, mutant tRNAs that decode the UAG stop (classically called the “amber” codon) are being aminoacylated with nonstandard amino acids.Then these tRNAs direct the incorporation of the novel amino acid into a nascent protein if its gene has been mutated to contain an amber codon at a specific site within its coding sequence. At this time, only a few nonstandard amino acids may be incorporated in this way, but it is anticipated that it will soon be possible to label proteins with a large variety of amino acids containing affinity tags, fluors, or reactive groups (4). Discussion of related topics can be found in “Nonsense suppression,” “Suppressor mutation,” and “Suppressor tRNA.”.

References

1. E. Kubli (1986) Trends Genet. 2, 204–209. 

2. F. H. C. Crick, L. Barnett, S. Brenner, and R. J. Watts-Tobin (1961) Nature 192, 1227–1232. 

3. D. R. Helinsky and C. Yanofsky (1963) J. Biol. Chem. 238, 1043–1048. 

4. E. J. Murgola (1994) in tRNA: Structure, Biosynthesis, and Function (D. Söll and U. L. RajBhandary, eds.), American Society for Microbiology Press, Washington, DC, pp. 491–509.