title: Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme.
abstract: We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA's topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes.
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Department of Chemical Engineering, ∥Department of Physics, Stanford University , Stanford, California 94305, United States.
authors.forename: Xuesong
authors.initials: X
authors.lastname: Shi
authors.forename: Namita
authors.initials: N
authors.lastname: Bisaria
authors.forename: Tara L
authors.initials: TL
authors.lastname: Benz-Moy
authors.forename: Steve
authors.initials: S
authors.lastname: Bonilla
authors.forename: Dmitri S
authors.initials: DS
authors.lastname: Pavlichin
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D016337:RNA, Catalytic / Q000737:chemistry*; D013768:Tetrahymena / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D016337:RNA, Catalytic
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