Date Published: March 22, 2019
Publisher: Public Library of Science
Author(s): Benjamin M. Spector, Michael E. Turek, David H. Price, Sukesh R. Bhaumik.
Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.
Pol II transcription is a tightly regulated process that is, in part, controlled by the phosphorylation status of the CTD of the largest Pol II subunit. The human CTD is composed of 52 repeats of the consensus heptad sequence Y1S2P3T4S5P6S7 and each hydroxyl containing residue is specifically phosphorylated at certain stages of transcription by particular CTD kinases. The pattern of CTD phosphorylation generated by these kinases is further altered by CTD phosphatases to create a dynamic signal that recruits factors needed at different stages of transcription and RNA processing [1–3].
We have examined association of Ssu72 with Pol II during early stages of transcription. Ssu72 becomes associated with Pol II during PIC formation, remains associated during very early elongation, and then is either inactivated or removed as complexes enter the paused state (Fig 6). We demonstrated that Ssu72 is capable of dephosphorylating early transcription complexes and uncovered a surprising transcript length-dependence for the phosphatase activity. Through unique usage of the EC-EMSA, we were able to remove the effects of other CTD phosphatases to specifically measure the rate and efficiency of Ssu72 activity. Initial experiments with this assay revealed that EECs only have EDTA-resistant phosphatase activity associated and that only addbacks containing Ssu72 could rescue this EDTA-resistant phosphatase activity. Phosphatase activity remained associated with EECs after washing with up to 200 mM KCl and antibody supershifts were used to correlate physical association of Ssu72 protein with phosphatase activity. Isolation of PICs prior to initiation did not eliminate the phosphatase activity on EECs. This indicates that the phosphatase becomes associated with the transcription complex during PIC assembly. Chasing EECs in the presence of extract resulted in the loss of phosphatase activity once transcripts exceeded 28 nt in length. Removal of unbound factors or factors loosely associated with the PIC abrogated the distinct transcript length restriction. Therefore, during the transition between initiation and pausing, the action of unknown factor(s) eliminates phosphatase activity on transcription complexes (Fig 6). Overall, we confirmed an earlier study showing that a CTD phosphatase was associated with EECs , identified it as Ssu72, demonstrated association of phosphatase activity with PICs, and discovered that Ssu72 activity was lost as nascent transcripts were extended beyond 28 nt.