C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats.

Published

Journal Article

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-Pro3-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-mRNA 3'-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-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Full Text

Duke Authors

Cited Authors

  • Jones, JC; Phatnani, HP; Haystead, TA; MacDonald, JA; Alam, SM; Greenleaf, AL

Published Date

  • June 11, 2004

Published In

Volume / Issue

  • 279 / 24

Start / End Page

  • 24957 - 24964

PubMed ID

  • 15047695

Pubmed Central ID

  • 15047695

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M402218200

Language

  • eng

Conference Location

  • United States