H/ACA ribonucleoprotein particles(RNPs),the most complicated RNA pseudouridylases,use H/ACA guide RNA for substrate capture and four proteins(Cbf5,Nop10,L7Ae and Gar1)for pseudouridylation.Gar1 has been shown to facilitate product release and promote reaction.However,in single turnover reactions,removal of Gar1 dramatically inhibits the reaction rates.The mechanistic details of catalysis promotion of Gar1 remain unclear.Using fluorescence anisotropy kinetics measurement,we find that Gar1 does not affect the activation energy of modification by H/ACA RNP.Structure-directed mutagenesis combined with molecular dynamics simulations demonstrate that Gar1 can optimally position the substrate RNA especially the target uridine close to the catalytic D85 in the active site through the interaction of Gar1 with N-terminal segment of the thumb loop.Conversion of uridine to pseudouridine causes greatly conformation change of the target nucleotide in the active site,which forms the structural basis for discrimination of substrate and product by H/ACA RNP.