Original Data of Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II by Sun and Fisher

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by protein phosphorylation. Previously, two regions phosphorylated by cyclin-dependent kinase 9 (CDK9) in elongation factor SPT5—the linker between KOWx-4 and KOW5 domains and carboxy-terminal repeat (CTR) 1—were implicated in promoter-proximal pausing and termination, respectively. Here we show that phosphorylations in the linker, CTR1 and a third region, CTR2, coordinately control pause release, elongation speed and termination. Pausing was unaffected or increased by mutations preventing CTR1 or CTR2 phosphorylation, respectively, but attenuated when both CTRs were mutated. Whereas loss of CTR1 phosphorylation slowed elongation and repressed nascent transcription, simultaneous CTR2 mutation partially reversed both effects. Nevertheless, mutating both CTRs had additive effects on splicing, termination, steady-state mRNA levels and cell proliferation. Therefore, tripartite SPT5 phosphorylation times pause release and tunes RNAPII elongation rate to ensure productive transcription and cell viability. This file contains the unprocessed and uncompressed imaging data.