Structural mechanism of substrate RNA recruitment in H/ACA RNA-guided pseudouridine synthase.
H/ACA RNAs form ribonucleoprotein complex (RNP) with proteins Cbf5, Nop10, L7Ae, and Gar1 and guide site-specific conversion of uridine into pseudouridine in cellular RNAs. The crystal structures of H/ACA RNP with substrate bound at the active site cleft reveal that the substrate is recruited through sequence-specific pairing with guide RNA and essential protein contacts. Substrate binding leads to a reorganization of a preset pseudouridylation pocket and an adaptive movement of the PUA domain and the lower stem of the H/ACA RNA. Moreover, a thumb loop flips from the Gar1-bound state in the substrate-free RNP structure to tightly associate with the substrate. Mutagenesis and enzyme kinetics analysis suggest a critical role of Gar1 and the thumb in substrate turnover, particularly in product release. Comparison with tRNA Psi55 synthase TruB reveals the structural conservation and adaptation between an RNA-guided and stand-alone pseudouridine synthase and provides insight into the guide-independent activity of Cbf5.
Duan
Jingqi
J
College of Life Sciences, Peking University, Beijing, China.
Li
Ling
L
Lu
Jing
J
Wang
Wei
W
Ye
Keqiong
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D020718:Ribonucleoproteins, Small Nucleolar; D012313:RNA; D019751:Intramolecular Transferases; C096995:pseudouridine synthases