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A two-stage molecular model for control of mini-F replication
Abstract:
It is known that mini-F replication requires production of a 29,000-Da protein, protein E, and origin of replication sequences mapping around 45. kb. Further, control of replication is determined by two genes, copA and copB. In the present work a description is given of the cloning of an F restriction fragment containing the amino terminal portion of the protein E gene, repE, and associate promoter activity. It is shown that expression of this promoter is negatively regulated in trans by sequences taken from the F replication region of copA+ plasmids. However, the same sequences taken from six different copA- plasmids failed to repress expression of the promoter. Since prior studies have shown that copA+ determines a repressor of replication, it is now suggested that the above results are an accounting of where this repressor works. A hypothesis is also proposed to explain control of F replication by the copA and copB regulatory genes.
Insights
This study identifies the mechanism by which the copA gene regulates mini-F plasmid replication. It demonstrates that copA repressor protein controls the expression of the replication initiator protein E gene (repE).
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Mini-F plasmid replication requires the 29,000-Da protein E and specific origin sequences.
- Replication control is governed by two genes: copA and copB.
Purpose of the Study:
- To clone and characterize the repE gene and its promoter.
- To investigate the regulatory role of copA and copB in mini-F replication.
Main Methods:
- Cloning of an F restriction fragment containing the amino-terminal portion of the protein E gene (repE) and its promoter.
- Assessing promoter activity and its regulation by F replication region sequences.
Main Results:
- Expression of the repE promoter is negatively regulated in trans by sequences from copA+ plasmids.
- Sequences from copA- plasmids failed to repress repE promoter expression.
- This suggests copA+ encodes a repressor that acts on the repE promoter.
Conclusions:
- The copA gene product directly represses the expression of the replication initiator protein E gene (repE).
- A hypothesis is proposed for the control of F replication by copA and copB regulatory genes.