title: An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions.
abstract: Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such "off-pathway" species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2'- and 3'-deoxy (-H) and -amino (-NH(2)) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3'-OH making a nonproductive interaction with an active site metal ion termed MA and with the adjacent 2'-OH making no interaction. Upon S binding, a rearrangement occurs that allows both -OH groups to contact a different active site metal ion, termed M(C), to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA's difficulty in specifying a unique conformation and highlighting Nature's potential to use local transitions of RNA in complex function.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Raghuvir N
authors.initials: RN
authors.lastname: Sengupta
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands.
authors.forename: Sabine N S
authors.initials: SN
authors.lastname: Van Schie
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: George
authors.initials: G
authors.lastname: Giambaşu
authors.affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Dai
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Yesselman
authors.affiliation: Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: Darrin
authors.initials: D
authors.lastname: York
authors.affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Piccirilli
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305, USA Department of Chemical Engineering, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California 94305, USA Department of Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California 94305, USA Department of Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, California 94305, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D009690:Nucleic Acid Conformation; D015347:RNA Probes; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013768:Tetrahymena / Q000201:enzymology*
chemical_list: D015347:RNA Probes; D016337:RNA, Catalytic
keywords: RNA catalysis; active site; conformational change; metal ion; noncoding RNA