Multiple kinases modify RNA Polymerase II (Pol II) and its associated pausing and elongation factors to regulate Pol II transcription and transcription-coupled mRNA processing1,2. The conserved Cdk9 kinase is essential for regulated eukaryotic transcription3, but its mechanistic role remains incompletely understood. Here, we use altered-specificity kinase mutations and highly-specific inhibitors in fission yeast, Schizosaccharomyces pombe to examine the role of Cdk9, and related Cdk7 and Cdk12 kinases, on transcription at base-pair resolution using Precision Run-On sequencing (PRO-seq). Within a minute, Cdk9 inhibition causes a dramatic reduction in the phosphorylation of Pol II-associated factor, Spt5. The effects of Cdk9 inhibition on transcription are the more severe than inhibition of Cdk7 and Cdk12 and result in a shift of Pol II towards the transcription start site (TSS). A kinetic time course of Cdk9 inhibition reveals that early transcribing Pol II is the most compromised, with a measured rate of only ~400 bp/min, while Pol II that is already well into the gene continues rapidly to the end of genes with a rate > 1 kb/min. Our results indicate that while Pol II in S. pombe can escape promoter-proximal pausing in the absence of Cdk9 activity, it is impaired in elongation, suggesting the existence of a conserved global regulatory checkpoint that requires Cdk9 kinase activity.
Beyond the canonical role of TFIIB in eukaryotic transcription
