CHARACTERIZATION OF THE RESIDUES PHOSPHORYLATED IN-VITRO BY DIFFERENTC-TERMINAL DOMAIN KINASES

The C-terminal part of the largest subunit of eukaryotic RNA polymeras e II is composed solely of the highly repeated consensus sequence Tyr( 1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). This domain, called the C-terminal domain (CTD), is phosphorylated mostly at serine residues d uring transcription initiation, but the precise role of this phosphory lation remains controversial, Several protein kinases are able to phos phorylate this sequence in vitro, The aim of this work was to define t he positions of the amino acids phosphorylated by four of these CTD ki nases (transcription factor (TF) IIH-kinase, DNA-dependent protein kin ase, and the mitogen-activated protein kinases ERK1 and ERK2) and to c ompare the specificity of these different protein kinases, We show tha t TFIIH kinase and the mitogen-activated protein kinases phosphorylate only serine 5 of the CTD sequence, whereas DNA-dependent protein kina se phosphorylates serines 2 and 7, Among the different CTD kinases, on ly TFIIH kinase is appreciably more active on two repeats of the conse nsus sequence than on one motif, These in vitro results can provide so me clues to the nature of the protein kinases responsible for the in v ivo phosphorylation of the RNA polymerase CTD. In particular, the rati o of phosphorylated serine to threonine observed in vivo cannot be exp lained if TFIIH kinase is the only protein kinase involved in the phos phorylation of the CTD.