Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription

Abstract

Edited by John M. Denu The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser 5 (pSer 5) is gradually dephosphorylated by CTD phosphatases, whereas Ser 2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer 5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer 2 and pSer 5 CTDs. We found that Ssu72 dephosphorylates pSer 5 effectively but only has low activities toward pSer 7 and pSer 2. The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ␤-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr 1 next to Ser 2 , prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr 1 further prohibited Ssu72 binding to pSer 2 and thereby prevented untimely Ser 2 dephosphorylation. Our results reveal critical roles for Tyr 1 in differentiating the phosphorylation states of Ser 2 / Ser 5 of CTD in RNA polymerase II that occur at different stages of transcription.

Ssu72 Dual-Specific Protein Phosphatase: From Gene to Diseases

“…In particular, it can interact with the CTD Ser5 kinase Kin28 to dephosphorylate CTD Ser5P and function in transcription initiation [ 8 ]. In addition, Ssu72 associates with the CTD phosphatase Fcp1 and Ctk1, which phosphorylates Ser2P in yeast, although some studies have revealed that Ssu72 has no Ser2P phosphatase activity because the Tyr1 residue does not fit into the tight β-turn of Ssu72 [ 8 , 26 , 27 , 28 ]. Ssu72 depletion can induce Fcp1 inactivation and early Ser2 phosphorylation by Ctk1, indicating that Ssu72 can tightly coordinate with various kinases and phosphatases during the transcription cycle [ 29 ].…”

What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domainviamass spectrometry

“…For example, MS analysis revealed the capability of FCP1 (the phosphatase responsible for recycling RNAP II) to dephosphorylate most of the CTD residues. 52 MS was likewise used to show that Ssu72 (a phosphatase responsible for dephosphorylating the Ser5 conserved across yeast and human) exhibits surprisingly high specificity towards Ser5 but not Ser2 even though both serines are part of an SP motif ( Fig. 3a ).…”

“…3a ). 52 These conclusions prompted investigators to take a closer look into the crystal structures of Ssu72, ultimately revealing that the narrow and deep active site imposes steric restrictions which regulate its specificity limited to Ser5 but not Ser2. 52 …”

A combinatorial view of old and new RNA polymerase II modifications

“…Molecular modeling studies predict that the presence of cis-proline significantly disrupts the active site of SCP1, thus making SCP1 preferentially act on serine residues proximal to transprolines [154]. Ssu72 is ubiquitously expressed and acts only on CTD repeats when prolines are in cis conformation [153,155,157].…”