Murray J A, Scarpa M, Rossi N, Cesareni G
EMBL, Heidelberg, FRG.
EMBO J. 1987 Dec 20;6(13):4205-12. doi: 10.1002/j.1460-2075.1987.tb02768.x.
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.
酵母的内源性2μm质粒编码一种位点特异性重组酶FLP，它可导致质粒拷贝数的扩增。利用从染色体构建体中过表达2μm质粒蛋白的菌株来破坏基因产物的正常平衡，我们在此表明，拷贝数是通过调节FLP的转录水平来控制的。FLP的表达在100倍的范围内受到质粒编码的REP1和REP2蛋白共同作用的负调控，这两种蛋白在质粒分配中也发挥作用。我们还表明，第四个质粒开放阅读框D的产物通过解除REP1和REP2引起的抑制作用来增加FLP的表达。这是该基因功能的首次证明，我们将其称为RAF。RAF的转录也受到REP1和REP2共同作用的抑制，但完全抑制所需的水平高于抑制FLP所需的水平。因此，拷贝数通过REP1-REP2浓度受到负调控，这既是通过直接抑制FLP，也是通过控制正向元件即抗阻遏物RAF间接实现的。我们提出，这些拮抗调节机制放大了拷贝数小幅下降所产生的信号。
