Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.
Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits.
复制标题
体内和体外酵母 DNA 依赖性 RNA 聚合酶的磷酸化。
DOI：
10.1016/s0021-9258(17)40474-1
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
William J. Rutter
中科院分区：
文献类型：
--
作者：
Graeme I. Bell;Pablo Valenzuela;William J. Rutter
酵母 DNA 依赖性 RNA 聚合酶 I、II 和 III 在体内被磷酸化。酵母细胞在 32Pi 中连续生长，并通过新程序分离 RNA 聚合酶，该程序允许从少量（35 至 60 g）细胞中同时纯化这些酶。每种 RNA 聚合酶均被磷酸化。鉴定了以下磷酸化聚合酶多肽：185,000、44,000、36,000、24,000和20,000道尔顿的聚合酶I亚基； 24,000 道尔顿的聚合酶 II 亚基；以及 24,000 和 20,000 道尔顿的聚合酶 III 亚基。掺入的 32P 对酸稳定，但对碱不稳定。纯化的[32P]聚合酶 I 部分酸水解后，鉴定出磷酸丝氨酸和磷酸苏氨酸。在部分分离过程中与聚合酶 I 共纯化的酵母蛋白激酶被部分纯化和表征。该蛋白激酶磷酸化体内磷酸化的纯化聚合酶亚基，此外还有 48,000 道尔顿的聚合酶 I 亚基和 33,500 道尔顿的聚合酶 II 亚基。在使用天然酵母 DNA 作为模板的简单测定中，用该蛋白激酶对纯化的酶进行磷酸化对聚合酶活性没有实质性影响。纯化的聚合酶 I 与酸性或碱性磷酸酶预温育对聚合酶活性也没有可检测到的影响。
Yeast DNA-dependent RNA polymerases I, II, and III are phosphorylated in vivo. Yeast cells were grown continuously in 32Pi and the RNA polymerases were isolated by a new procedure which allows the simultaneous purification of these enzymes from small quantities (35 to 60 g) of cells. Each of the RNA polymerases was phosphorylated. The following phosphorylated polymerase polypeptides were identified: polymerase I subunits of 185,000, 44,000, 36,000, 24,000, and 20,000 daltons; a polymerase II subunit of 24,000 daltons; and polymerase III subunits of 24,000 and 20,000 daltons. The incorporated 32P was acid-stable but base-labile. Phosphoserine and phosphothreonine were identified after partial acid hydrolysis of purified [32P]polymerase I. A yeast protein kinase that co-purifies with polymerase I during part of the isolation procedure was partially purified and characterized. This protein kinase phosphorylates the subunits of the purified polymerases that are phosphorylated in vivo and, in addition, a polymerase I subunit of 48,000 daltons and a polymerase II subunit of 33,500 daltons. Phosphorylation of the purified enzymes with this protein kinase had no substantial effect on polymerase activity in simple assays using native yeast DNA as a template. Preincubation of purified polymerase I with acid or alkaline phosphatase also had no detectable effect on polymerase activity.