T7 protein synthesis in F-factor-containing cells: evidence for an episomally induced impairment of translation and relation to an alteration in membrane permeability.
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+ spisome and two sipsomal 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+) celld retain the ability to bind Fmet defective T7-infected PIFA+, B+ rigosomes 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 of pifB- prevents the loss of ATP from the cells after T7 infection.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000581:Amidohydrolases / Q000096:biosynthesis; D002463:Cell Membrane Permeability; D002474:Cell-Free System; D003090:Coliphages / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*; D003227:Conjugation, Genetic; D004267:DNA Viruses; D004926:Escherichia coli / Q000378:metabolism*; D005144:F Factor; D009154:Mutation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis*; D014779:Virus Replication
D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000581:Amidohydrolases