Cdk-related kinase 9 regulates RNA polymerase II mediated transcription in Toxoplasma gondii

Cyclin-dependent kinases are an essential part of eukaryotic transcriptional machinery. In Apicomplexan parasites, the role and relevance of the kinases in the multistep process of transcription seeks more attention given the absence of full repertoire of canonical Cdks and cognate cyclin partners. In this study, we functionally characterize T. gondii Cdk-related kinase 9 (TgCrk9) showing maximal homology to eukaryotic Cdk9. An uncanonical cyclin, TgCyclin L, colocalizes with TgCrk9 in the parasite nucleus and co-immunoprecipitate, could activate the kinase in-vitro. We identify two threonines in conserved T-loop domain of TgCrk9 that are important for its activity. The activated TgCrk9 phosphorylates C-terminal domain (CTD) of TgRpb1, the largest subunit of RNA polymerase II highlighting its role in transcription. Selective chemical inhibition of TgCrk9 affected serine 2 phosphorylation in the heptapeptide repeats of TgRpb1-CTD towards 3' end of genes consistent with a possible role in transcription elongation. Interestingly, TgCrk9 kinase activity is regulated by the upstream TgCrk7 based CAK complex. TgCrk9 was found to functionally complement the role of its yeast counterpart Bur1 establishing its role as an important transcriptional kinase. In this study, we provide robust evidence that TgCrk9 is an important part of transcription machinery regulating gene expression in T. gondii.