Graham I R, Chambers A
Department of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
Curr Genet. 1996 Jul 31;30(2):93-100. doi: 10.1007/s002940050106.
The promoter of the RAP1 gene contains four potential binding sites for Rap1p, located between the UAS and the RNA initiation site. We have confirmed that three of these sites are recognised by Rap1p in vitro. Different combinations of the three sites were then mutated to abolish Rap1p binding, and the effect of these mutations on promoter activity was determined. When all three Rap1p sites were mutated, the activity of the promoter increased by about 130%, indicating that at least one of the sites is a negative element. Analysis of promoters with different combinations of the mutant sites revealed that the 5'-most site (A) is the principal target for repression. To test the involvement of Rap1p in controlling RAP1 expression, we have measured transcription of the chromosomal RAP1 gene in a RAP1 wild-type strain and two strains containing rap1ts mutations. At a semi-permissive temperature, the RAP1 promoter was more active in the rap1ts strains than in the RAP1 wild-type strain, suggesting that expression of the chromosomal RAP1 gene is greater when the activity of Rap1p in the cell is compromised. The activities of the wild-type promoter, and the promoter with mutations in the three Rap1p-binding sites, were compared in sir1, sir2, sir3 and sir4 mutant strains. In each case, the mutated promoter was significantly more active than the wild-type promoter, implying that the repression mechanism is not dependent on any one of the SIR gene products.
RAP1基因的启动子含有四个Rap1p潜在结合位点，位于上游激活序列（UAS）和RNA起始位点之间。我们已证实其中三个位点在体外可被Rap1p识别。然后将这三个位点的不同组合进行突变以消除Rap1p结合，并测定这些突变对启动子活性的影响。当所有三个Rap1p位点都发生突变时，启动子活性增加了约130%，表明至少其中一个位点是负调控元件。对具有不同突变位点组合的启动子进行分析发现，最靠近5'端的位点（A）是抑制作用的主要靶点。为了测试Rap1p在控制RAP1表达中的作用，我们测定了RAP1野生型菌株和两个含有rap1ts突变的菌株中染色体RAP1基因的转录情况。在半允许温度下，RAP1启动子在rap1ts菌株中比在RAP1野生型菌株中更活跃，这表明当细胞中Rap1p的活性受损时，染色体RAP1基因的表达更高。在sir1、sir2、sir3和sir4突变菌株中比较了野生型启动子和三个Rap1p结合位点发生突变的启动子的活性。在每种情况下，突变后的启动子都比野生型启动子活性显著更高，这意味着抑制机制不依赖于任何一种SIR基因产物。
