Determinants for dephosphorylation of the RNA polymerase II C-terminal domain by Scp1.
Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.
Zhang
Yan
Y
Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California 92037.
Kim
Youngjun
Y
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093.
Genoud
Nicolas
N
Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037.
Gao
Jianmin
J
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037.
Kelly
Jeffery W
JW
Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037.
Pfaff
Samuel L
SL
Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037.
Gill
Gordon N
GN
Department of Medicine, University of California, San Diego, La Jolla, California 92093; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093.
Dixon
Jack E
JE
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093; Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093.
Noel
Joseph P
JP
Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California 92037. Electronic address: noel@salk.edu.
D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry*; D020079:Terminal Repeat Sequences
D009687:Nuclear Proteins; D010455:Peptides; D012319:RNA Polymerase II; C413483:CTDSP1 protein, human; D010749:Phosphoprotein Phosphatases