Volume 1

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    Suppressor Mutations in the Anticodon Suppressor tRNAs with changed codon recognitions might result from either of two classes of mutation. The su gene might code for a modifying enzyme—for example a methylase—so that the mutant enzyme activity fails to make some modification essential for proper codon recognition. Such mutants should be recessive. (It is of course possible that mutation in an activating enzyme might alter rather than abolish its activity, but this is less likely to occur.) Alternatively, the su locus may comprise a structural gene for the tRNA, in which case the mutation usually directly changes one of the bases in the anticodon. These mutants should be dominant. The existence of allelic amber and ochre suppressors which may be converted by single base changes suggests that these su loci at least represent structural genes. It is difficult to isolate suppressor tRNAs from cells in amounts sufficient for sequencing because they are usually derived from minor tRNA species which are only synthesised to a small extent. The su3 suppressor locus, however, is located close to the attachment site of phage </>80; Smith et al. (1966) there fore used lysogenization of prophage 080 followed by induction to obtain transducing phages; these (f)80dsu3 phages are defective (indicated by the d) because they contain certain bacterial genes—including the su3 locus—in place of phage functions which are needed to complete the reproductive cycle of the phage. Two genes which code for the su3~ species of tRNA are located close together —probably adjacent—on the bacterial chromosome; both these genes are gained by the transducing phage. Bacteria with the su3 + characteristic are mutated in one of these genes but retain a wild type su3~ copy of the other; the structure of the phages prepared from su3 + bacteria is therefore (j)80dsu3 + - su3~, although—when bacteria were thought to contain only one locus coding for su3 tRNA—it was originally designated as (f)80dsu3 + . When the (f)SOdsu3 + su3~ phage infects bacteria, large amounts of suppressor su3 + tRNA and wild type su3~ tRNA are synthesised from the two bacterial genes which it carries. When cells are infected with the phage, lysis is delayed because it is defective in late functions but the reproductive cycle can be even further prolonged by the addition of chloramphenicol to block protein syn thesis. Transfer RNA can then be extracted from the infected cells with phenol. Comparison of the accepting levels of tRNA preparations for tyrosine and other amino acids in cells infected with (/)80dsu3 + su3~ shows that the relative propor tion of tyrosine tRNA is greatly increased by infection whereas the accepting capacity for other amino acids remains unaltered. In cells infected with </>80d su3 + su3~, the tyrosine accepting tRNA responds both to its usual codons UAU and UAC and also to the amber triplet UAG. When similar experiments are performed with the phage </>80d su3~su3~ prepared from su3~ bacteria, the same increase in tyrosine accepting activity is found, but the extra tRNA molecules continue to respond only to the normal codons UAU and UAC.