title: Structural principles of RNA catalysis in a 2'-5' lariat-forming ribozyme.
abstract: RNA-catalyzed lariat formation is present in both eukaryotes and prokaryotes. To date we lack structural insights into the catalytic mechanism of lariat-forming ribozymes. Here, we study an artificial 2'-5' AG1 lariat-forming ribozyme that shares the sequence specificity of lariat formation with the pre-mRNA splicing reaction. Using NMR, we solve the structure of the inactive state of the ribozyme in the absence of magnesium. The reaction center 5'-guanosine appears to be part of a helix with an exceptionally widened major groove, while the lariat-forming A48 is looped out at the apex of a pseudoknot. The model of the active state built by mutational analysis, molecular modeling, and small-angle X-ray scattering suggests that A48 is recognized by a conserved adenosine, juxtaposed to the 5'-guanosine in one base-pair step distance, while the G1-N7 coordinates a magnesium ion essential for the activation of the nucleophile. Our findings offer implications for lariat formation in RNA enzymes including the mechanism of the recognition of the branch-site adenosine.
authors.affiliation: Structural and Computational Biology Unit, EMBL, Meyerhofstraße 1, D-69117 Heidelberg, Germany. teresa.carlomagno@embl.de
authors.forename: Teresa
authors.initials: T
authors.lastname: Carlomagno
authors.forename: Irene
authors.initials: I
authors.lastname: Amata
authors.forename: Luca
authors.initials: L
authors.lastname: Codutti
authors.forename: Melanie
authors.initials: M
authors.lastname: Falb
authors.forename: Jörg
authors.initials: J
authors.lastname: Fohrer
authors.forename: Pawel
authors.initials: P
authors.lastname: Masiewicz
authors.forename: Bernd
authors.initials: B
authors.lastname: Simon
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; D008274:Magnesium