Mutations which affect the structure and activity of colicin E3.

Abstract

Among 69 ColE3 mutant plasmids selected on the basis of their inability to produce an active colicin, seven (cop-1 to cop-7) were found to bear a mutation affecting the structural gene for colicin. Three of these (cop-1, cop-2 and cop-3) lead to the production of an inactive colicin molecule which has the same molecular weight as wild-type colicin E3 (67,000). These three inactive colicins are still able to interact with the outer membrane receptor. The cop-1 protein retained the ability to inhibit protein synthesis in vitro and therefore seems specifically affected in it ability to penetrate the cell envelope. The cop-2 and cop-3 proteins lost the ability to inhibit protein synthesis in vitro, and activity which is normally associated with the C-terminal part of the colicin molecule. On the basis of this and further evidence, it is suggested that the cop-2 and cop-3 mutations affect the structure of the C-terminal part of the colicin molecule. The other four mutations (cop-4 to cop-7) lead to the production of colicin-related polypeptides of lower molecular weight (29,000 to 45,000) which display a reaction of partial immunological identity with wild-type colicin. These four polypeptides are unable to interact with the cell surface receptor. Three of these mutants are shown to carry a nonsense mutation.