Liesen T, Hollenberg C P, Heinisch J J
Institut für Mikrobiologie, Hainrich-Heine-Universität Düsseldorf, Germany.
Mol Microbiol. 1996 Aug;21(3):621-32. doi: 10.1111/j.1365-2958.1996.tb02570.x.
Yeast pyruvate decarboxylase (Pdc) catalyses the reaction at the branch-point of fermentation and respiration. In this work we have investigated the mechanisms of its transcriptional regulation in response to glucose and the non-fermentable carbon source ethanol. For this purpose we studied the function of different promoter fragments of PDC1, encoding the major pyruvate decarboxylase enzyme in wild-type cells, in the basal CYC1 promoter context. Thus, we identified a sequence mediating the response to ethanol and provide evidence showing that transcription of PDC1 is controlled by ethanol repression rather than by glucose induction. Furthermore, we showed that the same sequence is responsible for an autoregulatory process, leading to increased transcription from both the PDC1 and the PDC5 promoters, in strains in which the genomic copy of PDC1 is deleted. In addition, we have confirmed the role of Rap1 binding and have demonstrated that the Gcr1 protein also acts in transcriptional activation. DNA-protein interactions at the consensus Rap1-binding site and the newly identified ethanol-repression sequence (5'-AAATGCATA-3', termed 'ERA') were investigated by gel-shift and footprint analyses. Both DNA-binding activities were found in extracts from cells grown in media containing glucose or ethanol as the carbon source, indicating that the capacity to bind is not altered by the carbon source used.
酵母丙酮酸脱羧酶（Pdc）催化发酵和呼吸分支点处的反应。在这项工作中，我们研究了其响应葡萄糖和不可发酵碳源乙醇的转录调控机制。为此，我们在基础CYC1启动子背景下研究了编码野生型细胞中主要丙酮酸脱羧酶的PDC1不同启动子片段的功能。因此，我们鉴定了一个介导对乙醇响应的序列，并提供证据表明PDC1的转录受乙醇阻遏而非葡萄糖诱导的控制。此外，我们表明相同的序列负责一个自调控过程，在PDC1基因组拷贝被缺失的菌株中，导致PDC1和PDC5启动子的转录增加。另外，我们证实了Rap1结合的作用，并证明Gcr1蛋白也在转录激活中起作用。通过凝胶迁移和足迹分析研究了共有Rap1结合位点和新鉴定的乙醇阻遏序列（5'-AAATGCATA-3'，称为“ERA”）处的DNA-蛋白质相互作用。在以葡萄糖或乙醇作为碳源的培养基中生长的细胞提取物中均发现了这两种DNA结合活性，表明结合能力不会因所用碳源而改变。
