Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.
Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.
复制标题
酿酒酵母脯氨酸利用途径 PUT3 转录激活因子的功能分析。
DOI：
10.1093/genetics/142.4.1069
复制
发表时间：
1996
期刊：
影响因子：
3.3
通讯作者：
Brandriss,MC
中科院分区：
文献类型：
--
作者：
desEtages,SA;Falvey,DA;Reece,RJ;Brandriss,MC
当生长培养基中缺乏优选的氮源时，脯氨酸可以作为酿酒酵母的氮源。 PUT3 是脯氨酸利用途径的激活剂，是编码将脯氨酸转化为谷氨酸的酶的基因转录所必需的。 PUT3 是一种 979 个氨基酸的蛋白质，它组成性结合其靶基因启动子中的短 DNA 序列，但在缺乏脯氨酸诱导和存在首选氮源的情况下不会激活其表达。为了了解 PUT3 如何从非活动状态转换为活动状态，对其功能域进行了剖析。生化和分子测试、结构域交换实验以及激活子组成型和激活子缺陷型突变蛋白的分析表明，PUT3 是二聚体，并通过其带负电荷的羧基末端激活转录，该末端似乎不包含脯氨酸响应结构域。在许多真菌激活剂中发现的保守中心结构域的突变会干扰激活，但不会影响 DNA 结合或蛋白质稳定性。中央结构域突变的基因内抑制子已被分离和分析。
Proline can serve as a nitrogen source for the yeastSaccharomyces cerevisiaewhen preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence in the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding or protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.