Ssu72 phosphatase-dependent erasure of phospho-Ser7 marks on the RNA polymerase II C-terminal domain is essential for viability and transcription termination.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) serves an important role in coordinating stage-specific recruitment and release of cellular machines during transcription. Dynamic placement and removal of phosphorylation marks on different residues of a repeating heptapeptide (YSPTSPS) of the CTD underlies the engagement of relevant cellular machinery. Whereas sequential placement of phosphorylation marks is well explored, genome-wide engagement of phosphatases that remove these CTD marks is poorly understood. In particular, identifying the enzyme that erases phospho-Ser7 (Ser7-P) marks is especially important, because we find that substituting this residue with a glutamate, a phospho-mimic, is lethal. Our observations implicate Ssu72 as a Ser7-P phosphatase. We report that removal of all phospho-CTD marks during transcription termination is mechanistically coupled. An inability to remove these marks prevents Pol II from terminating efficiently and will likely impede subsequent assembly into the pre-initiation complex.