Kinetic and thermodynamic characterization of the reaction pathway of box H/ACA RNA-guided pseudouridine formation

Xinxing Yang, Jingqi Duan, Shuang Li, Peng Wang, Shoucai Ma, Keqiong Ye, Xin Sheng Zhao

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)10925-10936
Number of pages12
JournalNucleic acids research
Volume40
Issue number21
DOIs
StatePublished - Nov 2012

ASJC Scopus subject areas

  • Genetics

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