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Positive and negative roles of an initiator protein at an origin of replication
Abstract:
The properties of mutants in the pir gene of plasmid R6K have suggested that the pi protein plays a dual role; it is required for replication to occur and also plays a role in the negative control of the plasmid copy number. In our present study, we have found that the pi level in cell extracts of Escherichia coli strains containing R6K derivatives is surprisingly high (approximately equal to 10(4) dimers per cell) and that this level is not altered in cells carrying high copy number pir mutants. The wild-type and a high copy mutant (Cos405) pir gene were inserted downstream of promoters of different strengths to measure the copy number of an R6K gamma replicon as a function of a 1000-fold range of intracellular pi concentrations. The data demonstrate that reducing the intracellular level of pi to 5% of its normal value can result in a substantial increase in copy number of a gamma origin replicon and that a pi level less than 1% of normal is still permissive for replication. Conversely, increasing the pi level even a few-fold above normal results in a marked inhibition of replication of plasmids containing a single, two, or all three of the R6K origins (alpha, beta, and gamma). We have also shown that the replication inhibition mediated by excess pi is greatly reduced by the pir405 Cos mutation. These results demonstrate that the total level of pi protein is not rate-limiting for a gamma replicon. We have also determined the sensitivity of the pir gene promoter to a wide range of pi concentrations. The activity of this promoter is stimulated by very low pi levels and is almost entirely inhibited when the protein is overproduced 2-fold.
Insights
The pir protein regulates R6K plasmid replication and copy number. Lowering pir levels boosts replication, while excess pir inhibits it, with a specific mutation reducing this inhibition.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The pir gene product of plasmid R6K is known to be essential for replication and involved in copy number control.
- Previous studies suggested a dual role for the pir protein in R6K plasmid replication and negative control.
Purpose of the Study:
- To investigate the quantitative relationship between pir protein levels and R6K plasmid replication.
- To determine the effect of varying intracellular pir concentrations on plasmid copy number and replication efficiency.
Main Methods:
- Cloning wild-type and mutant pir genes under inducible promoters of varying strengths.
- Measuring R6K plasmid copy number across a 1000-fold range of intracellular pir concentrations in E. coli.
- Assessing the impact of pir protein levels on plasmids with different R6K origins (alpha, beta, gamma).
Main Results:
- Intracellular pir protein levels in E. coli are high (approx. 10^4 dimers/cell) and unaffected by high copy number pir mutants.
- Reducing pir levels to 5% of normal significantly increases copy number of gamma origin replicons; levels below 1% remain permissive for replication.
- Overproducing pir protein even a few-fold inhibits replication of plasmids with one, two, or all three R6K origins.
- The pir405 mutation significantly reduces replication inhibition caused by excess pir protein.
- The pir gene promoter is stimulated by low pir levels and inhibited by overproduction (2-fold).
Conclusions:
- The total level of pir protein is not rate-limiting for gamma replicon replication.
- Pir protein concentration critically influences R6K plasmid replication and copy number, acting as both an initiator and a repressor.
- The pir405 mutation alters the regulatory properties of the pir protein, affecting its interaction with replication origins or its own promoter.