Giesman D, Best L, Tatchell K
Graduate Group of Molecular Biology and Genetics, University of Pennsylvania, Philadelphia 19104.
Mol Cell Biol. 1991 Feb;11(2):1069-79. doi: 10.1128/mcb.11.2.1069-1079.1991.
The RAP1 gene of Saccharomyces cerevisiae encodes an abundant DNA-binding protein, also known as GRF1, TBA, or TUF, that binds to many sites in the yeast genome in vitro. These sites define a consensus sequence, [sequence: see text], and deletion analyses of genes that contain this sequence have implicated the involvement of RAP1 in numerous cellular processes, including gene activation and repression. The MAT alpha locus, required for determination of the alpha cell type in yeast cells, contains a RAP1 binding site; this site coincides with the MAT alpha upstream activating sequence (UAS) and is necessary for expression of the two genes encoded by the MAT alpha locus, MAT alpha 1 and MAT alpha 2. We show that the MAT alpha UAS is sufficient to activate transcription from a promoterless gene fusion of the yeast CYC1 upstream region and the lacZ gene. Constructs containing only the MAT alpha UAS generated elevated levels of beta-galactosidase activity which were indistinguishable from those of constructs containing the entire MAT alpha intergenic region. Further, the MAT alpha UAS has an intrinsic polarity of transcriptional activation; transcription of CYC1-lacZ was six- to sevenfold higher when the UAS was oriented in the direction normally associated with MAT alpha 2 transcription. Point mutations in the MAT alpha UAS that reduce MAT alpha expression three- to fivefold resulted in a bi-mating phenotype, while a mutation that reduced MAT alpha expression still further resulted in an a-mating phenotype. We isolated plasmids from a high-copy-number yeast library that suppressed the bi-mating defect of point mutations in the MAT alpha UAS, and the most effective dosage suppressor contained the gene encoding RAP1. A temperature-sensitive rap1 mutant bi-mates at the semipermissive temperature. Double mutants at rap1 and mat alpha mate exclusively as a cells, at all temperatures, and do not express detectable levels of MAT alpha RNA. These data provide evidence that the RAP1 gene product functions at the MAT alpha UAS in vivo.
酿酒酵母的RAP1基因编码一种丰富的DNA结合蛋白，也称为GRF1、TBA或TUF，该蛋白在体外可与酵母基因组中的多个位点结合。这些位点定义了一个共有序列，[序列：见正文]，对包含该序列的基因进行缺失分析表明，RAP1参与了许多细胞过程，包括基因激活和抑制。酵母细胞中决定α细胞类型所需的MATα位点包含一个RAP1结合位点；该位点与MATα上游激活序列（UAS）重合，是MATα位点编码的两个基因MATα1和MATα2表达所必需的。我们表明，MATα UAS足以激活酵母CYC1上游区域与lacZ基因的无启动子基因融合的转录。仅包含MATα UAS的构建体产生的β-半乳糖苷酶活性水平升高，与包含整个MATα基因间区域的构建体的活性水平没有区别。此外，MATα UAS具有转录激活的内在极性；当UAS以通常与MATα2转录相关的方向定向时，CYC1-lacZ的转录高出六至七倍。MATα UAS中的点突变使MATα表达降低三至五倍，导致双交配表型，而进一步降低MATα表达的突变导致a交配表型。我们从高拷贝数酵母文库中分离出质粒，这些质粒抑制了MATα UAS中点突变的双交配缺陷，最有效的剂量抑制子包含编码RAP1的基因。温度敏感型rap1突变体在半允许温度下双交配。rap1和matα的双突变体在所有温度下都仅作为a细胞交配，并且不表达可检测水平的MATα RNA。这些数据提供了证据，表明RAP1基因产物在体内的MATα UAS处发挥作用。
