Biochemistry - The Chemical Reactions of Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
Genetic Methods
The Nature of Suppressor Genes

What chemical processes underlie the suppression of one mutation by another located at a different site within the chromosome? There is no straightforward answer to this question. Rarely is a mutation suppressed by another located within the same Gene. Such an effect may be termed intragenic complementation. Suppose a mutation leads to an amino acid substitution that disrupts structural stability or protein function. It is possible that a mutation at another site, involving a residue that interacts with the substituted amino acid, alters The Nature of the interaction between the two residues, thereby restoring the functional activity of the protein. For example, if the side chain of the first amino acid is small and is replaced As a result of a mutation by a longer side chain, a second mutation that reduces the size of another side chain may enable the resulting protein to fold and function like a normal protein. Such a case was discovered among Tryptophan synthetase mutants [144]. Mutants of this protein in which Gly-211 was replaced by Glu, or Tyr-175 by Cys, synthesized inactive Enzymes, whereas the double mutant—that is, the mutant carrying both of these substitutions—synthesized active tryptophan synthetase. It is believed that in most cases of intragenic suppression, alterations occur in the subunit interactions of Oligomeric Proteins.

Suppressor genes that suppress A large number of various Mutations leading to premature chain termination are the most thoroughly studied. The Chemical Nature of these genes has been only partially elucidated through experiments involving The transfer of the suppressor gene supF (su3) into bacteriophage DNA. It turned out that this DNA specifically hybridizes with minor species of Tyrosine tRNA (tyrosine tRNA_I). Subsequent research showed that suIII serves as the structural gene for a minor tyrosine tRNA in which the normal anticodon GUA is replaced by the triplet CUA, which is capable of pairing with the termination codon UAG (the amber codon). As a result, tyrosine is incorporated at the site of the termination signal caused by amber mutations. Somewhat surprisingly, the tRNA that prevents chain termination does not hinder the Synthesis of Other essential proteins in the bacterial Cell. However, the efficiency of suppression usually does not exceed 30%. Consequently, the synthesis of many polypeptide chains terminates normally. If the presence of two chain termination signals is a general property of genes, the Synthesis of the majority of proteins in the presence of suppressor tRNA should terminate normally. Nevertheless, premature chain termination caused by amber mutations will be partially inhibited, allowing The Cell to synthesize The amount of the missing enzyme necessary for survival. The nucleotide sequences of supF-tRNA and its longer precursor are shown in Fig. 15-9.

A number of other suppressor genes, which are structural genes for specific tRNAs, have also been identified [140]. Recently, for instance, a frameshift suppressor mutation was discovered in the Glycine tRNA gene of Salmonella typhimurium [145]. In this tRNA, the anticodon position contains four bases, CCCC, instead of the usual triplet CCC. This is the only known tRNA in which the anticodon loop contains eight unpaired NUCLEOTIDES instead of the usual seven.

Suppressor genes are found not only in Bacteria. For example, the vermilion mutation in Drosophila is suppressed by a mutation in the tryptophan tRNA gene [140]. The vermilion mutation results in a failure to synthesize brown eye pigment, which is explained by the inactivation of tryptophan oxygenase [Equation (10-45)]. It was found that the tryptophan oxygenase of the vermilion mutant is inhibited by one of the two tryptophan tRNAs, specifically tRNA₂^Trp. During the suppressor mutation, the tRNA is altered in such a way that this inhibition is relieved [140].

Miller, Lu, and their coworkers [145a, b] successfully utilized suppressor mutations to generate about 300 mutant types of the E. coli lac repressor protein. In The First stage, amber mutations were introduced at approximately 80 positions within the gene. Next, for cloning purposes, the mutant genes were transferred into episomes (see the following section). These virus-like episomes were then used to infect five bacterial strains carrying suppressor mutations that allowed the reading of the UAG (termination) codon to result in the incorporation of various Amino Acids into the protein. Large quantities of mutant forms of the lac repressor were isolated from these infected bacteria. It turned out that many mutations located near the N-terminus affect repressor binding to DNA, whereas mutations located in the central region affect binding to the inducer.