Wang J, Sirenko O, Needleman R
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
J Biol Chem. 1997 Feb 14;272(7):4613-22. doi: 10.1074/jbc.272.7.4613.
Mig1p inhibits gene expression in glucose by binding the Cyc8p (Ssn6p)-Tup1p repressor to the promoter of glucose-repressible genes. While the binding properties of Mig1p have been studied in vitro and the ability of Mig1p-Cyc8p (Ssn6p)-Tup1p to repress has been studied in vivo, no experiments have measured the effect of a carbon source on the in vivo binding of Mig1p or the effect of bound MIg1p on activator occupancy of the upstream activation sequence (UAS). To obtain this information, we used genomic footprinting to investigate glucose repression of MAL62, a gene that is also regulated by the Mal63p activator. These experiments show that two interrelated mechanisms are involved in the glucose repression of MAL62: 1) competition between the Mal63p activator and Mig1p for DNA binding and 2) modulation of Mig1p binding by the carbon source. Mig1p affects basal MAL62 expression in the absence of Mal63p by binding to a site in the MAL62 promoter and affects Mal63p-dependent synthesis by also inhibiting the access of Mal63p to site 1 in the UASMAL. The binding of Mig1p is increased in glucose and decreased in nonrepressing sugars, but the increased binding in glucose is not due to an increase in the levels of Mig1p.
Mig1p通过将Cyc8p（Ssn6p）-Tup1p阻遏物与葡萄糖可阻遏基因的启动子结合，来抑制葡萄糖存在时的基因表达。虽然已在体外研究了Mig1p的结合特性，且在体内研究了Mig1p-Cyc8p（Ssn6p）-Tup1p的阻遏能力，但尚无实验测量碳源对Mig1p体内结合的影响，或结合的Mig1p对上游激活序列（UAS）上激活剂占据情况的影响。为获取这些信息，我们使用基因组足迹法研究了MAL62的葡萄糖阻遏作用，MAL62是一个也受Mal63p激活剂调控的基因。这些实验表明，MAL62的葡萄糖阻遏涉及两种相互关联的机制：1）Mal63p激活剂与Mig1p之间对DNA结合的竞争；2）碳源对Mig1p结合的调节。Mig1p通过与MAL62启动子中的一个位点结合，在没有Mal63p的情况下影响MAL62的基础表达，并通过抑制Mal63p进入UASMAL中的位点1，来影响Mal63p依赖性合成。Mig1p的结合在葡萄糖中增加，在非阻遏性糖类中减少，但葡萄糖中结合增加并非由于Mig1p水平升高。
