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.

DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

“…As each frayed base pair is successively captured, the loss of base stacking is expected to weaken the adjacent base pair, accelerating its fraying and therefore accelerating unwinding [43] . In a conceptually analogous manner, when a DEAD-box protein captures a helix from a structured RNA, it will not only destabilize tertiary structure by preventing reformation of tertiary contacts by the captured helix, but it will also weaken additional tertiary contacts within the folded RNA if they form cooperatively [44] – [46] . Thus, despite its passive nature, this helix capture mechanism is expected to accelerate the kinetics of large-scale tertiary unfolding of structured RNAs.…”

Quantitative tests of a reconstitution model for RNA folding thermodynamics and kinetics

“…This analysis extracts transition probabilities between each state while taking into account the noise observed in each intensity channel. Criteria for trace selection were described previously (72); briefly, traces were selected based on exhibiting all of the following: (i) single-step photobleaching, (ii) anticorrelated donor and acceptor channels, (iii) total intensity consistent with a single molecule, and (iv) stable total intensity.…”

Probing the kinetic and thermodynamic consequences of the tetraloop/tetraloop receptor monovalent ion-binding site in P4–P6 RNA by smFRET

“…For P4–P6 folding, a simple two-state model was used. smFRET imaging, trace selection and processing was done as described previously [49,50], using a custom total internal reflection (TIRF) setup with image acquisition by Andor iXon Ultra camera and the Nikon Elements software. All smFRET data were taken with a 20 ms exposure time, an average single-to-noise ratio of 2.0 (defined in [47]), in 1.8 M monovalent-chloride salt, 100 mM Na–MOPS, pH 7.0, 0.1 mM EDTA solutions at 25°C, with an oxygen scavenging system of 2 mg/ml glucose, 1.8 mM Trolox, 100 units/ml glucose oxidase and 1000 units/ml catalase.…”