Replication of plasmid RK2 in vitro by a DNA-membrane complex: evidence for initiation of replication and its coupling to transcription and translation.
The following results with an in vitro replication system utilizing a plasmid RK2 DNA-membrane complex indicate that the essential trfA-encoded replication protein of RK2 is present and active in the complex. (i) A complex extracted from a conditional replication mutant of RK2, which contains a temperature-sensitive mutation in trfA, displayed extensive DNA synthesis at the permissive temperature but little activity at the restrictive temperature. A control wild-type RK2 complex showed no inhibition of DNA synthesis at the restrictive temperature. (ii) Analysis of plasmid-encoded proteins revealed that the trfA-specified replication protein and other proteins which may be involved in the replication and maintenance of RK2 are located physically in the complex. Semiconservative plasmid DNA replication by the DNA-membrane complex was indicated by density shift experiments; DNA synthesized in the presence of a heavy-density precursor banded primarily in a heavier-density area of a neutral CsCl density gradient and consisted mostly of heavy- and light-density single-stranded DNA as determined by alkaline CsCl density gradient centrifugation. Plasmid RK2 DNA replication by the DNA-membrane complex appears to be coupled to transcription and translation as indicated by the following results: the inhibitory effects of chloramphenicol on both DNA and protein synthesis by the complex; the stimulation of replication by components normally required for protein synthesis (tRNA and all the common amino acids); the synthesis of RNA and protein by the complex; and the synthesis of specific RK2-encoded proteins.
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D004265:DNA Helicases; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D014176:Protein Biosynthesis; D011815:R Factors; D012329:RNA, Bacterial / Q000096:biosynthesis; D013696:Temperature; D015534:Trans-Activators; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases