Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription.
Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription.
复制标题
DOI：
10.1016/s0021-9258(19)38281-x
复制
发表时间：
1990-08
期刊：
影响因子：
--
通讯作者：
P. Laybourn;M. Dahmus
中科院分区：
文献类型：
--
作者：
P. Laybourn;M. Dahmus
哺乳动物RNA聚合酶II的最大亚基在其C末端含有一个不寻常的结构域，由7个氨基酸共同序列Tyr-Ser-Pro-Thr-Ser-Pro-Ser的多个串联重复组成。该结构域在RNA聚合酶IIA中不磷酸化，在RNA聚合酶IIO中广泛磷酸化。为了研究C末端结构域的作用及其磷酸化的功能意义，我们跟踪了磷酸化水平的变化，作为RNA聚合酶II在转录周期中位置的函数。用~(32)P标记的RNA聚合酶IIA与其形成复合物，并通过凝胶过滤从游离的聚合酶中分离出来。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定RNA聚合酶II最大亚基的磷酸化状态。结果表明，RNA聚合酶IIA与模板承诺的复合体相互作用，形成稳定的预引发复合体。与这种复合体相关的RNA聚合酶IIA在ATP存在下转化为RNA聚合酶IIO，然后形成第一个磷酸二酯键。此外，纯化的预引发复合体能够催化RNA聚合酶IIA向IIO的转化，这表明负责C-末端结构域磷酸化的蛋白激酶(S)是这种复合体的组成部分。与预引发复合体相关的RNA聚合酶II的磷酸化所需的ATP浓度比在溶液中将RNA聚合酶IIA转化为IIO所需的浓度低两到三个数量级。这些结果支持这样的观点，即RNA聚合酶亚基IIa的C-末端结构域的磷酸化发生在酶与启动子结合之后、转录启动之前。
The largest subunit of mammalian RNA polymerase II contains at its C terminus an unusual domain consisting of multiple tandem repeats of the seven-amino acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unphosphorylated in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. To investigate the role of the C-terminal domain and the functional significance of its phosphorylation, changes in the level of phosphorylation were followed as a function of the position of RNA polymerase II in the transcription cycle. Complexes were formed with 32P-labeled RNA polymerase IIA and separated from the free polymerase by gel filtration. The phosphorylation state of the RNA polymerase II largest subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that RNA polymerase IIA interacts with the template-committed complex to form a stable preinitiation complex. RNA polymerase IIA associated with such complexes is converted to RNA polymerase IIO in the presence of ATP prior to the formation of the first phosphodiester bond. Furthermore, the observation that purified preinitiation complexes can catalyze the conversion of RNA polymerase IIA to IIO indicates that the protein kinase(s) responsible for phosphorylation of the C-terminal domain is a component of such complexes. The concentration of ATP required for the phosphorylation of RNA polymerase II associated with the preinitiation complex is two to three orders of magnitude lower than that required for the conversion of RNA polymerase IIA to IIO free in solution. These results support the idea that phosphorylation of the C-terminal domain of RNA polymerase subunit IIa occurs subsequent to the association of enzyme with the promoter and prior to the initiation of transcription.