title: Accurate placement of substrate RNA by Gar1 in H/ACA RNA-guided pseudouridylation.
abstract: H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product.
authors.affiliation: College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.affiliation: College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China.
authors.forename: Lijiang
authors.initials: L
authors.lastname: Yang
authors.affiliation: College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China gaoyq@pku.edu.cn.
authors.forename: Yi Qin
authors.initials: YQ
authors.lastname: Gao
authors.affiliation: College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China zhaoxs@pku.edu.cn.
authors.forename: Xin Sheng
authors.initials: XS
authors.lastname: Zhao
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D019277:Entropy; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011560:Pseudouridine / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D020537:RNA, Small Nucleolar; D020718:Ribonucleoproteins, Small Nucleolar; D011560:Pseudouridine; D012313:RNA; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D014529:Uridine