Phosphorylation site specificity of CTD kinase I.

摘要

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of heptapeptides with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5 -Pro6-Ser7. The CTD is unique to RNA polymerase II, and it is essential for viability in all eukaryotes that have this domain. Phosphorylation of this domain correlates with polymerase activity. Specifically, phosphorylation of serines at position 5 is required for the transition from initiation to elongation, while Ser2 phosphorylation is detected on polymerases transcribing coding regions of genes.; Differentially phosphorylated forms of the CTD bind different sets of proteins that are involved in a spectrum of nuclear events including capping, splicing, 3' end formation and chromatin remodeling. A network of kinases and phosphatases combine to determine the phosphorylation pattern of the CTD throughout the transcription cycle. Our lab has focused on budding yeast CTD kinase I (CTDK-I). Genetics and biochemistry implicate this kinase in elongation, chromatin remodeling and 3' end formation. CTDK-I has been exploited as a tool in our lab to generate hyperphosphorylated CTD to use as a probe for interactants in the yeast proteome, but little was known about how this kinase phosphorylates the CTD.; We examined the specificity of CTDK-I and found that it phosphorylates CTD heptads that are already phosphorylated at Ser2 or Ser5 more efficiently than unphosphorylated heptads. CTDK-I phosphorylates Ser5 of substrates that are either unphosphorylated or phosphorylated at Ser2, but it phosphorylates Ser2 if Ser5 is already phosphorylated. We have identified an arginine residue in the T-loop of the catalytic subunit of CTDK-I that is an important determinant of substrate specificity and enzyme processivity. Finally, we compared the specificity of CTDK-I to P-TEFb, the proposed metazoan counterpart to CTDK-I. We found that although CTDK-I and P-TEFb have some differences, they share more similarities with each other than either shares with other CTD kinases. We propose a model wherein CTDK-I acts after the TFIIH-associated CTD kinase to generate CTD heptads that are phosphorylated at both Ser2 and Ser5, and we suggest that doubly-phosphorylated CTD repeats contribute importantly to elongation and associated functional properties of RNAPII.