Probing the Dynamics of the P1 Helix within the Tetrahymena Group I Intron

Abstract

RNA conformational transformations are integral to RNA's biological functions. Further, structured RNA molecules exist as a series of dynamic intermediates in the course of folding or complexation with proteins. Thus, an understanding of RNA folding and function will require deep and incisive understanding of its dynamic behavior. However, existing tools to investigate RNA dynamics are limited. Here we introduce a powerful fluorescence polarization anisotropy approach that utilizes a rare base analog that retains substantial fluorescence when incorporated into helices. We show that 6-methylisoxanthopterin (6-MI) can be used to follow the nanosecond dynamics of individual helices. We then use 6-MI to probe the dynamics of an individual helix, referred to as P1, within the 400nt Tetrahymena group I ribozyme. Comparisons of the dynamics of the P1 helix in wild type and mutant ribozymes and in model constructs reveal a highly immobilized docked state of the P1 helix, as expected, and a relatively mobile ‘open complex’ or undocked state. This latter result rules out a model in which slow docking of the P1 helix into its cognate tertiary interactions arises from a stable alternatively docked conformer. The results are consistent with a model in which stacking and tertiary interactions of the A3 tether connecting the P1 helix to the body of the ribozyme limit P1 mobility and slow its docking, and this model is supported by cross-linking results. The ability to isolate the nanosecond motions of individual helices within complex RNAs and RNA/protein complexes will be valuable in distinguishing between functional models and in discerning the fundamental behavior of important biological species.

A Single‐Stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme

“…[15] The EPR data obtained for Ç are in agreement with the fluorescence results. Analyses presented here further suggest that J1/2 modulates motional ordering of P1 in the 0.5–50 ns range.…”

Tightening of Active Site Interactions En Route to the Transition State Revealed by Single-Atom Substitution in the Guanosine-Binding Site of the Tetrahymena Group I Ribozyme

“…We monitored the reactions under conditions in which the oligonucleotide substrate (S) is already bound to the ribozyme in the so-called open complex [(E•S) o , Figure 1a], because the open complex gives a defined state that involves only base pairing of S with the ribozyme that is not expected to be affected by modifications in the guanosine binding site. 10,40,41 To favor the open complex, we used the oligonucleotide substrate -1r,dSA 5 (see General kinetic methods ), in which specific 2′-OH groups that stabilize the docked state are replaced by 2′-H groups.…”

RNA dynamics: perspectives from spin labels

“…It has been proposed that intrinsic dynamics of P1 in the open complex, which control the ability of P1 to sample different conformations (e.g., open & closed complex), may be one of the factors responsible for the observed slow docking rate. 50, 52, 53 However, virtually nothing is known about intrinsic dynamics of any individual states in large folded RNAs such as the Tetrahymena ribozyme. The SDSL studies described above was the first example in which intrinsic dynamics of P1 in the nanosecond regime were probed experimentally in the context of the entire ribozyme, and the data revealed that intrinsic dynamics are modulated by structure (i.e., J1/2) and may influence function (i.e., substrate binding affinity and cleavage fidelity).…”