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. 1976 Jan;17(1):94–105. doi: 10.1128/jvi.17.1.94-105.1976

T7 Protein Synthesis in F-Factor-Containing Cells: Evidence for an Episomally Induced Impairment of Translation and Its Relation to an Alteration in Membrane Permeability

Daphne D Blumberg 1, Cheryl T Mabie 1, Michael H Malamy 1
PMCID: PMC515392  PMID: 1107598

Abstract

T7 infection of F-factor-containing PIFA+,B+ cells is abortive. In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized. A crucial question is whether the failure of T7 to develop in PIFA+,B+ cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability. We have examined the effects of the wild-type PIFA+,B+ episome and two episomal mutations (pifA− and pifB−) on in vitro translation and membrane permeability. In vivo the episomal mutations allow partial or complete T7 development to occur. We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA+,B+ cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA. In addition, ribosomes from T7-infected PIFA+,B+ cells are defective in their ability to bind Fmet tRNAf in response to natural mRNA. By contrast, cell-free extracts from T7-infected pifA− (PIFA−,B+) cells retain the ability to bind Fmet tRNAf and to translate natural and synthetic mRNA at normal rates. The defective T7-infected PIFA+,B+ ribosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA+,B+ or T7-infected PIFA−,B+ cells. Despite the differences in translational capacity of these extracts, both T7-infected PIFA+,B+ and PIFA−,B+ cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant. Mutation of the episome to pfiB− prevents the loss of ATP from the cells after T7 infection.

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Selected References

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