The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.
The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.
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酿酒酵母 PUT3 激活蛋白与脯氨酸特异性上游激活序列相关。
DOI：
10.1128/mcb.9.11.4706-4712.1989
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发表时间：
1989
影响因子：
5.3
通讯作者：
Brandriss,MC
中科院分区：
文献类型：
--
作者：
Siddiqui,AH;Brandriss,MC
编码酿酒酵母利用脯氨酸途径酶的PUT1和PUT2基因是由Pro诱导的，并由PUT3基因的产物激活。PUT1启动子中的两个上游激活序列(UASs)与PUT2UAS同源。对这两个PUT1UASs的缺失分析表明，它们在功能上是独立的，在产生最高水平的基因表达方面是相加的。ConensusPUTUAS是一个21个碱基对的部分回文序列，体内需要这两个基因的诱导。凝胶迁移率改变分析的结果表明，脯氨酸特异的UAS是蛋白质因子的结合部位。在体外，携带PUT3基因的单个基因组拷贝的酵母菌株和克隆在2μm质粒上的PUT3基因的酵母菌株的粗提液中可以观察到复合体的形成，并且这种结合是剂量依赖的。在携带aput3突变的菌株的提取物中没有观察到DNA结合活性，aput3突变导致了不可诱导的(Put-)表型或该基因的缺失。在一株携带PUT3等位基因的菌株的提取物中观察到野生型复合体的形成，这导致了结构性(Put+)表型。携带PUT3-lacZ基因融合的菌株的提取物形成了两个迁移率比野生型复合体慢的复合体。我们得出结论，PUT3产物要么是DNA结合蛋白，要么是识别PUT1和PUT2 UASs的DNA结合复合体的一部分。在有或没有Pro的情况下，在菌株的提取物中观察到结合，证明了DNA-蛋白质相互作用的结构性。
ThePUT1andPUT2genes encoding the enzymes of the proline utilization pathway ofSaccharomyces cerevisiaeare induced by proline and activated by the product of thePUT3gene. Two upstream activation sequences (UASs) in thePUT1promoter were identified by homology to thePUT2UAS. Deletion analysis of the twoPUT1UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensusPUTUAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of thePUT3gene or the clonedPUT3gene on a 2μm plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either aput3mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele ofPUT3that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying aPUT3-lacZgene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that thePUT3product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of bothPUT1andPUT2.Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.