title: Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation.
abstract: The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Department of Chemical Biology, College of Chemistry and Molecular Engineering, and Biodynamic Optical Imaging Center, Peking University, Beijing 100871, China.
authors.forename: Xinxing
authors.initials: X
authors.lastname: Yang
authors.forename: Jingqi
authors.initials: J
authors.lastname: Duan
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Shoucai
authors.initials: S
authors.lastname: Ma
authors.forename: Keqiong
authors.initials: K
authors.lastname: Ye
authors.forename: Xin Sheng
authors.initials: XS
authors.lastname: Zhao
mesh_terms: D020134:Catalytic Domain; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011560:Pseudouridine / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D014529:Uridine / Q000378:metabolism; D058727:RNA, Small Untranslated
chemical_list: D020537:RNA, Small Nucleolar; D020718:Ribonucleoproteins, Small Nucleolar; D011560:Pseudouridine; D012313:RNA; D019751:Intramolecular Transferases; C096995:pseudouridine synthases; D014529:Uridine; D058727:RNA, Small Untranslated