Regulation of ColE1 Plasmid DNA Replication in Escherichia coli

Citation

Moser, David Randall (1984) Regulation of ColE1 Plasmid DNA Replication in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/bf0f-4378. https://resolver.caltech.edu/CaltechTHESIS:10302018-183325232

Abstract

A number of plasmid copy-number mutants have been characterized in order to gain insight into the mechanism by which initiation of plasmid DNA synthesis is regulated. One nutant, pFHllB, which has a 12-fold higher copy number than the homologous wildtype plasmid, RSF1050, contains 16 additional base pairs within a region of the plasmid that has been shown to be essa1tial for norrral replication. The insertion nutation lies approximately 500 base tairs upstream of the origin of DNA synthesis within the coding region of a small untranslated RNA, RNA I. The copy-number nutation is recessive and can be complemented both in cis and in trans by the wild-type RNA I gene. The results of the complementation studies reveal that replication of the ColE1-type plasmids is regulated by a mechanism of negative control in which RNA I functions as a repressor of plasmid DNA synthesis.

The target of RNA I inhibition is believed to be near the 5' end of a second plasmid transcript, RNA II. RNA II transcription begins 555 base pairs upstream of the replication origin and terminates several hundred base pairs downstream of the origin. RNA II can be processed by RNase H to generate a primer for the initiation of DNA synthesis by DNA polymerase I. Purified RNA I has been shown to inhibit primer formation in vitro. Dominant high copy-number mutants of a specially-constructed plasmid, pDM247, contain single base-pair changes within a seven base-pair sequence of the RNA II coding region. The altered nucleotides in RNA II are located within the single-stranded loop of a prominent hairpin structure that can form near the 5' end of the transcript. A romplenentary hairpin structure exists in RNA I. A model for RNA I-RNA II interaction involving base pairing of loop structures has been proposed.

A single base-pair mutation has been identified in a plasmid which has a temperature-sensitive high ropy-number phenotype. The ts mutant exhibits normal copy control in cells growing at the permissive temperature of 30°C but replicates uncontrollably in cells shifted to the nonpermissive temperature, 37°C. Runaway replication of plasmid DNA is lethal to the host cells within 3-4 hours after the temperature shift. The orp mutation (for over-replication) affects both the RNA II transcript and the promoter for RNA I. RNA I promoter-galK fusion studies indicate that the mutation does not create a temperature-sensitive promoter. It has been proposed that the orp mutation creates a thermosensitive secondary structure in RNA II. The breakdown of this structure at the higher temperature renders the plasmid insensitive to replication inhibition by RNA I. Secondary mutations which suppress the temperature-sensitive phenotype have been isolated. These mutations also affect nucleotides within RNA II.

A small plasmid-encoded polypeptide also plays a role in plasmid copy number control. Although this protein, referred to as the rop gene product, is not essential for regulation of plasmid DNA replication (the deletion of the rop gene causes only a three to five-fold increase in plasmid copy number), its presence in the cell can suppress lethal DNA over-replication of the temperature-sensitive copy number mutant. This suppression was used as the basis of a selection for rop insensitive mutants. The identification of point mutations within the RNA I coding region of these mutants is consistent with the hypothesis that the product of the rop gene modulates the interaction between RNA I and the primer precursor.

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