Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA.
Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.
Liang
Bo
B
Institute of Molecular Biophysics, Tallahassee, Florida, USA.
Zhou
Jing
J
Kahen
Elliot
E
Terns
Rebecca M
RM
Terns
Michael P
MP
Li
Hong
H
D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011560:Pseudouridine / Q000737:chemistry / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019843:Archaeal Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D011560:Pseudouridine; D012313:RNA; D019751:Intramolecular Transferases; C096995:pseudouridine synthases