SummarySequential modifications of the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) coordinate the stage-specific association and release of cellular machines during transcription. Here we examine the genome-wide distributions of the “early” (phospho-serine 5), “mid” (phospho-serine 7) and “late” (phospho-serine 2) CTD marks. We identify gene-class specific patterns and find widespread co-occurrence of the CTD marks. Contrary to its role in 3’ processing of non-coding RNA, the Ser7-P marks are placed early and retained until transcription termination at all Pol II-dependent genes. Chemical-genomic analysis reveals that the promoter-distal Ser7-P marks are not remnants of early phosphorylation, but are placed anew by the CTD kinase Bur1. Consistent with the ability of Bur1 to facilitate transcription elongation and suppress cryptic transcription, high levels of Ser7-P are observed at highly transcribed genes. We propose that Ser7-P could facilitate elongation and suppress cryptic transcription.

Background: Reversible phosphorylation of the RNA Polymerase II CTD coordinates co-transcriptional recruitment of factors. Results: Ssu72 is required for erasure of phospho-serine7, and it facilitates Fcp1-mediated phospho-serine2 removal. Conclusion: Removal of phospho-Ser7 mark plays a key role in the transcription cycle. Significance: Persistent negative charge at position 7 of the CTD renders cells non-viable, and Ssu72 plays a prominent role in removing phospho-Ser7.

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SUMMARY During transcription initiation, the TFIIH-kinase Kin28/Cdk7 marks RNA polymerase II (Pol II) by phosphorylating the C-terminal domain (CTD) of its largest subunit. Here we describe a structure-guided chemical approach to covalently and specifically inactivate Kin28 kinase activity in vivo. This method of irreversible inactivation recapitulates both the lethal phenotype and the key molecular signatures that result from genetically disrupting Kin28 function in vivo. Inactivating Kin28 impacts promoter release to differing degrees and reveals a “checkpoint” during the transition to productive elongation. While promoter-proximal pausing is not observed in budding yeast, inhibition of Kin28 attenuates elongation-licensing signals, resulting in Pol II accumulation at the +2 nucleosome and reduced transition to productive elongation. Furthermore, upon inhibition, global stabilization of mRNA masks different degrees of reduction in nascent transcription. This study resolves longstanding controversies on the role of Kin28 in transcription and provides a rational approach to irreversibly inhibit other kinases in vivo.