A novel chemical‐genomic approach for the dissection of the CTD code (940.2)

Abstract

The C‐terminal domain (CTD) of the largest subunit of RNA Pol II serves as a central hub for the regulation of transcription and its coordination with co‐transcriptional events. The CTD is composed of a highly conserved heptapeptide (Y1S2P3T4S5P6S7) repeated in tandem 26 times in the yeast Saccharomyces cerevisiae, and serves as a scaffold for an array of combinatorial post‐translational modifications which establish a CTD code. In transcription initiation, RNA Pol II and general transcription factors assemble at promoter regions prior to transcription elongation. The final factor to be recruited, TFIIH and its associated kinase, Kin28, phosphorylates the Ser5 and Ser7 residues of CTD. The co‐occurrence of Ser5 phosphorylation and transcription initiation led to the hypothesis that Ser5 phosphorylation by Kin28 is required for transcription initiation and RNA Pol II promoter release.Our initial chemical genomic analysis revealed that although Ser5 and Ser7 phosphorylation levels decrease upon inhibition of Kin28, RNA Pol II is still able to initiate transcription and engage in active elongation. However, because residual kinase activity may still be present once the inhibitor reaches equilibrium, we set out to re‐explore the role of Kin28 in general transcription through a more rigorous method. To compellingly dispel any doubt of our initial findings, we have applied a novel strategy in which we specifically and irreversibly block Kin28 kinase activity in vivo. Our results show that inhibition of Kin28 causes an arrest in cell growth consistent with its essential role in cellular processes. Additionally, chromatin immunoprecipitation and genome‐wide analyses reveal a dramatic decrease in Ser5 across the genome, while RNA Pol II remains at high levels across all transcribed genes. Our results suggest that in fact Kin28 is not required for general Pol II transcription and that targeted irreversible inhibition of kinases in vivo can be extended to other kinases.Grant Funding Source: Supported by NHGRI Training Grant to the Genomic Sciences Training Program (5T32HG002760)