Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation.
The largest subunit of eukaryotic RNA polymerase II (RNAP II) has a serine- and threonine-rich C-terminal domain (CTD) that may interact both with DNA and with the activating region of transcription factors. It has been proposed, in one model, that a protein kinase phosphorylates the promoter-associated CTD, facilitating the transition between promoter-binding and RNA-elongating forms of RNAP II. An immobilized template transcription system was used to test the predictions of this model directly. A protein kinase that phosphorylated the CTD at multiple sites was detected. This activity was tightly bound to the template, as evidenced by continued association after multiple rounds of washing. Phosphorylation was promoter sequence-dependent and exhibited the same nucleotide substrate specificity as the previously characterized ATP-requiring step in initiation. It was necessary for [gamma-32P]ATP and initiating rNTPs to be present simultaneously in the reaction in order to efficiently chase-radiolabel into elongating RNAP II-containing complexes, consistent with the idea that initiation and phosphorylation are temporally associated reactions.
Arias
J A
JA
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
Peterson
S R
SR
Dynan
W S
WS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D003902:Detergents; D004800:Enzymes, Immobilized; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic
D003902:Detergents; D004800:Enzymes, Immobilized; C025231:sarkosyl; D000255:Adenosine Triphosphate; D004247:DNA; D011494:Protein Kinases; D012319:RNA Polymerase II; D012521:Sarcosine