Updating the rna polymerase ctd
Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events. “Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat..” J Biol Chem, vol. For one well-known phospho CTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phospho CTD binding ability of Set2; surprisingly, Set2's role in damage resistance does not depend on its catalytic activity.
Using GST-CTD fusion protein substrates we find that CDK12/Cyclin K prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. “Expression, purification, and identification of associated proteins of the full-length h CDK12/Cyclin K complex..” J Biol Chem, vol. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phospho CTD-associating protein genes and DNA damage-resistance genes.
Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain.
THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II.
In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes.
Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death.