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Antagonistic controls regulate copy number of the yeast 2 mu plasmid
J A Murray1, M Scarpa, N Rossi
1EMBL, Heidelberg, FRG.
Abstract:
The endogenous 2 mu plasmid of yeast encodes a site-specific recombinase FLP that can cause an amplification of plasmid copy number. Using strains overexpressing 2 mu plasmid proteins from chromosomal constructs to disrupt the normal balance of gene products, we show here that copy number is controlled by regulating the transcript level of FLP. Expression of FLP is negatively regulated over a 100-fold range by the joint action of the plasmid-encoded REP1 and REP2 proteins, which also have a role in plasmid partition. We also show that the product of the fourth plasmid open reading frame D increases FLP expression by relieving the repression caused by REP1 and REP2. This is the first demonstration of a function for this gene, which we call RAF. The transcription of RAF is also repressed by REP1 and REP2 acting together, but requires a higher level for complete inhibition than that required to repress FLP. Copy number is therefore negatively regulated by REP1-REP2 concentration both by direct repression of FLP and indirectly, by control of the positive element, the anti-repressor RAF. We propose that these antagonistic regulatory mechanisms amplify the signal produced by a small fall in copy number.
Insights
Yeast 2-micron plasmid copy number is controlled by regulating FLP recombinase transcript levels. Repressor proteins REP1 and REP2, along with anti-repressor RAF, create a regulatory circuit that amplifies copy number signals.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Epigenetics
Background:
- The 2-micron plasmid in yeast is crucial for maintaining its episomal state and copy number.
- FLP recombinase is a key enzyme encoded by the 2-micron plasmid, responsible for copy number amplification.
- Understanding the regulatory mechanisms of plasmid replication is vital for genetic engineering and biotechnology.
Purpose of the Study:
- To elucidate the regulatory mechanisms controlling the transcript levels of the FLP recombinase gene.
- To identify the roles of plasmid-encoded proteins REP1, REP2, and the product of open reading frame D in regulating FLP expression.
- To characterize the function of the previously uncharacterized open reading frame D, designated RAF.
Main Methods:
- Utilized yeast strains with overexpressed 2-micron plasmid proteins from chromosomal constructs.
- Quantified transcript levels of FLP recombinase under various experimental conditions.
- Investigated the interaction between REP1, REP2, RAF, and FLP expression.
Main Results:
- FLP expression is negatively regulated by REP1 and REP2 proteins over a 100-fold range.
- The product of open reading frame D (RAF) acts as an anti-repressor, relieving REP1-REP2-mediated repression of FLP.
- RAF transcription is also repressed by REP1 and REP2, but requires higher levels for complete inhibition compared to FLP repression.
Conclusions:
- Yeast 2-micron plasmid copy number is finely tuned by a dual regulatory system involving repression and anti-repression.
- REP1 and REP2 proteins directly repress FLP expression and indirectly control it by regulating RAF, an anti-repressor.
- This antagonistic regulatory network amplifies responses to small fluctuations in plasmid copy number, ensuring stable propagation.