An unexpected binding mode for a Pol II CTD peptide phosphorylated at Ser7 in the active site of the CTD phosphatase Ssu72

Abstract

Ssu72, an RNA polymerase II C-terminal domain (CTD) phospho-Ser5 (pSer5) phosphatase, was recently reported to have pSer7 phosphatase activity as well. We report here the crystal structure of a ternary complex of the N-terminal domain of human symplekin, human Ssu72, and a 10-mer pSer7 CTD peptide. Surprisingly, the peptide is bound in the Ssu72 active site with its backbone running in the opposite direction compared with a pSer5 peptide. The pSer7 phosphatase activity of Ssu72 is~4000-fold lower than its pSer5 phosphatase activity toward a peptide substrate, consistent with the structural observations. Transcription of mRNA and noncoding RNA in eukaryotes is carried out by RNA polymerase II (Pol II), the activity of which is regulated in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit (Komarnitsky et al. 2000;Schroeder et al. 2000;Meinhart et al. 2005;Phatnani and Greenleaf 2006;Buratowski 2009;Kim et al. 2009Kim et al. , 2010Mayer et al. 2010;Tietjen et al. 2010). The CTD contains the consensus heptapeptide repeat Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 (YSPTSPS), and phosphorylation of Ser5 and Ser2 has long been recognized for its importance in transcription and coupled RNA processing events. Phosphorylation of Ser7 is involved in snRNA transcription and 39 end processing Egloff et al. 2007), and phosphorylation of Thr4 has been linked to histone mRNA 39 end processing (Hsin et al. 2011). CTD kinases and phosphatases control the phosphorylation state of the CTD and thereby regulate Pol II activity. For example, Fcp1 preferentially dephosphorylates pSer2 over pSer5 (Hausmann and Shuman 2002;Hausmann et al. 2004;Ghosh et al. 2008), while Ssu72 is a pSer5 phosphatase (Krishnamurthy et al. 2004;Hausmann et al. 2005). Both are essential for viability in yeast.Recently, it was reported that Ssu72 also has pSer7 phosphatase activity (Bataille et al. 2012;Zhang et al. 2012). However, such an activity for Ssu72 is unexpected from a structural perspective. Ssu72 recognizes the cis configuration of the pSer5-Pro6 peptide bond as a pSer5 phosphatase (Xiang et al. 2010;Werner-Allen et al. 2011). In comparison, pSer7 is followed by Tyr1 in the next repeat of the CTD (designated Tyr19). If Ssu72 were to bind the pSer5 and pSer7 substrates in the same way, the pSer7-Tyr19 peptide bond would need to be in the cis configuration, which is much less favorable energetically. Moreover, the bulkier Tyr side chain would be placed in the binding site for the Pro6 residue in the pSer5 substrate, which would clash with the enzyme. Results and DiscussionTo understand the structural basis for how Ssu72 functions as a pSer7 phosphatase, we determined the crystal structure at 2.2 Å resolution of a ternary complex of a human symplekin N-terminal domain (NTD, residues 30-360), human Ssu72 (C12S mutant), and a 10-mer CTD peptide phosphorylated at Ser7 (Ser2-Pro3-Thr4-Ser5-Pro6-pSer7-Tyr19-Ser29-Pro39-Thr49, with the prime indicating the next repeat of the CTD) (Fig. 1A). Symplekin is a scaffold protein in t...

The Ssu72 Phosphatase Mediates the RNA Polymerase II Initiation-Elongation Transition

“…Furthermore, this effect is evident from the promoter region of ADH1, as opposed to the gradual increase in the signal characteristic of Ser(P) 2 ChIPs in a WT strain (43). Because Ssu72 is specific for dephosphorylation of Ser(P) 5 and Ser(P) 7 (21,22,28), with no activity toward Ser(P) 2 (27,44), our results imply that Ssu72 indirectly regulates Ser 2 phosphorylation. Ssu72 could exert this effect by facilitating the recruitment and/or activation of the Ser(P) 2 phosphatase, Fcp1, at promoters.…”

“…Ssu72 is a CTD phosphatase specific for Ser(P) 5 and Ser(P) 7 and is essential for cell viability (21,22,27,28). Despite its essential role in CTD dephosphorylation, the specific role of Ssu72 in the transcription cycle remains unresolved.…”

“…Despite its essential role in CTD dephosphorylation, the specific role of Ssu72 in the transcription cycle remains unresolved. Intriguingly, the orientation of Ssu72 relative to the backbone polarity of the CTD is critical for substrate specificity: in one orientation, specificity is for Ser(P) 5 , whereas in the opposite orientation, specificity is for Ser(P) 7 , albeit with much lower activity (28). Ssu72 was first identified based on a genetic interaction with the general transcription factor TFIIB, an interaction that affects the accuracy of start site selection (29).…”

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

“…The three-dimensional structures of the first ∼400 amino acids of human CPSF73 and yeast CPSF100 (Mandel et al 2006) and the N-terminal ∼300 amino acids of Drosophila and human Symplekin were individually determined by X-ray crystallography (Kennedy et al 2009;Xiang et al 2010Xiang et al , 2012. These structures include important residues comprising the CPSF73 active site (Fig.…”

A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

“…Several phosphatases have been implicated to control S5P-CTD levels and coordinate these transitions at active genes. The Ssu72 CTD phosphatase selectively removes S5P and S7P without affecting S2P (Ganem et al 2003;Krishnamurthy et al 2004;Xiang et al 2012;Zhang et al 2012). In S. cerevisiae, Ssu72 predominantly occupies the 39 end of genes, where it promotes termination in conjunction with either CPF/ CPSF or Sen1:Nrd1:Nab3 complexes (Dichtl et al 2002;He et al 2003;Steinmetz and Brow 2003).…”