This chapter focuses on the purification process of protein kinases that phosphorylate the repetitive carboxyl-terminal domain (CTD) of eukaryotic RNA polymerase II. It presents an assay to detect CTD kinase and have purified enzymes which catalyze this reaction. This assay is used to isolate two protein kinase complexes, designated CTD kinases, which phosphorylate serine residues in the consensus heptapeptide repeat that composes the CTD. As the CTD is almost entirely composed of tandem heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, a few of the heptapeptide repeats would, in isolation, adopt a structure similar to heptapeptides within the CTD. A series of heptapeptides (hepta) are synthesized for use in two assay systems as potential protein kinase substrates, which demonstrates that peptides with three or fewer repeats are poor as substrates in these systems. Peptide substrates are prepared on an Applied Biosystems model 430A peptide synthesizer using tert-butyloxycarbonyl (t-Boc) chemistry. Following synthesis, peptides are cleaved and deblocked using HF, then chromatographed on a preparative Vydac reversed-phase C18 (1x25 cm) column. The principal assay used for the purification of CTD kinase employs the peptide Arg-hepta as the phosphate acceptor. After incubating the peptide in the presence of [γ-32P]ATP and enzyme, labeled peptide substrate is separated from unincorporated ATP by binding the arginine residues of the peptide to phosphocellulose paper, while removing the unbound ATP by washing. This assay can also be used to assess the relative affinity of the enzyme for various heptapeptides (or other substrates) by providing a nonbinding substrate (such as hepta-four) to serve as a competitor in the reaction.
ASJC Scopus subject areas
- Molecular Biology