Studies on a factor enhancing colicin E3 activity in vitro

Abstract

The mechanism of action of colicin E3 (E3) was investigated in an in vitro system. Purified ribosomes are less susceptible to E3 than crude or washed ribosomes. A factor was found in the supernatant fraction of normal Escherichia coli cells that stimulates inactivation of ribosomes by E3, and on addition of this factor, about one tenth as much E3 was required for inactivation of ribosomes. On heating a mixture of E3 and this factor above 60 degrees , the ribosome inactivating activity of E3 increased greatly, and an amount corresponding to 0.01 mug of E3 was sufficient to inactivate 1.0 A(260) unit of ribosomes completely. By this treatment bacteriocidal activity of E3 decreased considerably, as the ratio of the two activities of E3 (ribosome inactivating activity and bacteriocidal activity) increased to 6 x 10(4)-fold. It is evident that the two activities do not run in parallel. This heat-treated product cleaved 16S rRNA in the same way as E3. These results suggest that inactivation of ribosomes is not due to colicin molecules prepared by the standard procedure, but to a modified form of them.