Trigon S, Serizawa H, Conaway J W, Conaway R C, Jackson S P, Morange M
Ecole Normale Superieure, Unité de Génétique Moléculaire, 46, rue d'Ulm, 75230 Paris Cedex 05, France.
J Biol Chem. 1998 Mar 20;273(12):6769-75. doi: 10.1074/jbc.273.12.6769.
The C-terminal part of the largest subunit of eukaryotic RNA polymerase II is composed solely of the highly repeated consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This domain, called the C-terminal domain (CTD), is phosphorylated mostly at serine residues during transcription initiation, but the precise role of this phosphorylation remains controversial. Several protein kinases are able to phosphorylate this sequence in vitro. The aim of this work was to define the positions of the amino acids phosphorylated by four of these CTD kinases (transcription factor (TF) IIH-kinase, DNA-dependent protein kinase, and the mitogen-activated protein kinases ERK1 and ERK2) and to compare the specificity of these different protein kinases. We show that TFIIH kinase and the mitogen-activated protein kinases phosphorylate only serine 5 of the CTD sequence, whereas DNA-dependent protein kinase phosphorylates serines 2 and 7. Among the different CTD kinases, only TFIIH kinase is appreciably more active on two repeats of the consensus sequence than on one motif. These in vitro results can provide some clues to the nature of the protein kinases responsible for the in vivo phosphorylation of the RNA polymerase CTD. In particular, the ratio of phosphorylated serine to threonine observed in vivo cannot be explained if TFIIH kinase is the only protein kinase involved in the phosphorylation of the CTD.
真核生物RNA聚合酶II最大亚基的C末端部分仅由高度重复的共有序列Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7组成。这个结构域称为C末端结构域（CTD），在转录起始过程中主要在丝氨酸残基处被磷酸化，但其磷酸化的确切作用仍存在争议。几种蛋白激酶能够在体外使该序列磷酸化。这项工作的目的是确定这些CTD激酶中的四种（转录因子（TF）IIH激酶、DNA依赖性蛋白激酶以及丝裂原活化蛋白激酶ERK1和ERK2）所磷酸化的氨基酸位置，并比较这些不同蛋白激酶的特异性。我们发现TFIIH激酶和丝裂原活化蛋白激酶仅使CTD序列的丝氨酸5磷酸化，而DNA依赖性蛋白激酶使丝氨酸2和丝氨酸7磷酸化。在不同的CTD激酶中，只有TFIIH激酶对共有序列的两个重复序列的活性明显高于对一个基序的活性。这些体外实验结果可以为负责RNA聚合酶CTD体内磷酸化的蛋白激酶的性质提供一些线索。特别是，如果TFIIH激酶是唯一参与CTD磷酸化的蛋白激酶，那么在体内观察到的磷酸化丝氨酸与苏氨酸的比例就无法得到解释。
