novel modifications on C-terminal domain of RNA polymerase II can fine-tune the phosphatase activity of Ssu72

Abstract

The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats, through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intramolecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 does not dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 toward the CTD peptide by 4-fold. To our surprise, even though the intramolecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.

Figure 1

Mass spectrometric analysis of CTD peptides treated with Ssu72. a. 19-mer CTD peptide doubly phosphorylated at Thr4 and Ser5 was treated with Ssu72 for zero, 3.5 hours and overnight. LC-MS selected ion monitoring (SIM) chromatograms of the dephosphorylated Thr4/Ser5 precursor (left-most peak, monitoring m/z 1068 ± 25) and the singly dephosphorylated product (right-most peak, monitoring m/z 1028 ± 25) after exposure of diphosphoryl-peptide SPSYSPTSPSYSPpTpSPSYS to Ssu72. b. UVPD characterization to determine the retained site of phosphorylation on the product CTD peptide in (a) after overnight incubation with Ssu72. c. Analysis of Ser5/Ser7 dephosphorylation by CTD. Shown is a 193nm negative mode UVPD spectrum of the mass representing doubly phosphorylated CTD (phosphorylated Ser5/Ser7) treated by Ssu72. Shown in the inset are specific ions of interest that confirm the presence of the phosphoryl group at position Ser7 of the peptide.

Figure 2

Steady-state kinetics of Drosophila Ssu72-symplekin towards 19mer CTD peptides: SPSYSPTSPSYSPT(phos.)SPSYS (a) and SPSYSPTSPSYSP(phos.)T(phos.)SPSYS (b).

Figure 3

Structure of Drosophila melanogaster Ssu72-symplekin-CTD ternary complex in which the ligand is 19-mer CTD peptide with singly phosphorylated Ser5: a. The superimposed structures of Drosophila and human Ssu72-symplekin-CTD complexes. Symplekin and Ssu72 of Drosophila melanogaster are colored pale cyan and light pink, respectively. Symplekin and Ssu72 of human are colored yellow and green. CTD peptides bound by Drosophila and human Ssu72s were shown in stick model with the carbon atom colored yellow and green, respectively. b. Composite omit annealing map of electron density of CTD peptide of singly phosphorylated Ser5 (yellow) in Drosophila melanogaster Ssu72-symplekin complex, contoured at 3 σ. c. Overlay of the structures of Drosophila apo Ssu72 (wheat), Ssu72 in complex with the CTD (light pink), Ssu72 in complex with symplekin (grey), and Ssu72 in complex with symplekin and the CTD (green): a loop consisting of Ssu72 49–53 residues display a significant conformational change upon the binding of the CTD or symplekin. d. Interaction formed between Drosophila Ssu72 (violet) upon its association with template protein symplekin (lime green). Superimposition with apo Ssu72 (light blue) structure reveals a small conformational adjustment to accommodate symplekin binding.

Figure 4

Recognition of CTD by Drosophila melanogaster symplekin-Ssu72. a. Superimposition of singly phosphorylated Ser5 CTD peptide bound at the active site of Drosophila melanogaster Ssu72 (peptide carbon shown in yellow) and human Ssu72 (peptide carbon shown in green). b. The electrostatic surface potential of active site of Drosophila Ssu72, where blue indicates positive charge, red indicates negative charge, and white indicates hydrophobic regions. The surfaces were generated with the Adaptive Poisson–Boltzmann Solver (APBS) using the AMBER force field (APBS Tools 2.1 PyMoL plugin, M. G. Lerner). c. A hydrogen bond network formed around phosphate group of phos.Ser5 CTD peptide in active site of Ssu72. d. Key residues of Drosophila Ssu72 for CTD recognition. The amino acids of Ssu72 are colored pink while the carbon atoms of CTD peptide are shown in yellow.

Figure 5

Complex structure of Drosophila Ssu72-symplekin bound to doubly phosphorylated CTD peptide at Thr4/Ser5. a. Composite annealing omit map of CTD peptide with doubly phosphorylated Thr4 and Ser5, contoured at 3 σ. b. Overlay of structures of Drosophila Ssu72-symplekin in complex with 19-mer CTD peptides, singly phosphorylated Ser5 with carbon atoms colored yellow and doubly phosphorylated Thr4 and Ser5 with carbon shown in green. c. Model of combinatorial code of phosphorylation and isomerization. Thr4 phosphorylation occurring after the Ser2 phosphorylation will delay the dephosphorylation of Ser5.