C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase IIC-terminal domain repeats
C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase IIC-terminal domain repeats
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DOI：
10.1074/jbc.m402218200
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发表时间：
2004-06-11
影响因子：
4.8
通讯作者：
Greenleaf, AL
中科院分区：
文献类型：
--
作者：
Jones, JC;Phatnani, HP;Greenleaf, AL
RNA聚合酶II最大亚基的C末端重复结构域(CTD)由串联的七个重复序列组成，共有序列为Tyr1-Ser2-ro3-Thr4-Ser5-Pro6-Ser7。在酵母中，这个七肽序列重复大约26次，在转录过程中它变得过度磷酸化，主要是在丝氨酸2和5。一系列的激酶和磷酸酶结合在一起决定了CTD的磷酸化模式。我们试图确定酵母CTD激酶-I(CTDK-I)磷酸化的位置特异性，CTDK-I参与包括延伸和前mRNA3‘端形成在内的各种核过程。为此，我们鉴定了通常用于研究CTD磷酸化模式的单抗，并发现H5单抗与在Ser2和/或Ser5处磷酸化的CTD物种发生反应。因此，我们使用抗体不依赖的方法来研究CTDK-I，我们发现，如果CTD底物在Ser2处未磷酸化或预磷酸化，CTDK-I就会磷酸化CTD的Ser5。当Ser5已经被磷酸化时，CTDK-I将CTD的Ser2磷酸化。我们还观察到CTDK-I有效地产生双磷酸化的CTD重复序列；已经含有Ser2-PO4或Ser5-PO4的CTD底物比未磷酸化的CTD底物更容易被CTDK-I磷酸化。
The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-ro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO4 or Ser5-PO4 are more readily phosphorylated by CTDK-I than unphosphorylated CTD substrates.