Book of Abstracts: Albany 2003
June 17-21 2003
Single-molecule Fluorescence Microscopy of the Hairpin Ribozyme Reveals Elementary Principles of RNA Folding
RNA is a unique biopolymer that can carry both genetic information and catalytic function. Its secondary structure folds stably and independently, and assembles hierarchically into a modular tertiary structure. Studies of these folding events are key to understanding how RNA can fulfill its rich set of essential cellular tasks in regulation of gene expression, splicing, and translation. We have used single-molecule fluorescence resonance energy transfer (FRET) microscopy to monitor the kinetics of reversible folding from the undocked (secondary structured, catalytically inactive) to the docked (tertiary structured, catalytically active) conformation of the hairpin ribozyme (Fig. 1), a small RNA enzyme crucial for replication of the tobacco ringspot virus satellite RNA. We have found complex structural dynamics with four docked states of distinct stabilities and a strong memory effect where each molecule rarely switches between different docked states. We also have shown overall catalysis to be rate-limited by a combination of (un)folding and reversible chemistry equilibrium (Science 296, 1473-1476 (2002)).
Figure 1: Docking and undocking transitions of the hairpin ribozyme as observed by single-molecule FRET.
Examining the effects of mutations and solvent ionic strength on folding shows that the folding transition state is compact and electrostatically stabilized like the native state, but without substantial native tertiary contacts. The major barrier to folding is thus attributed to the rearrangement of secondary structure and/or the decrease in chain entropy, and is not electrostatic in origin. Our studies suggest that non-specific electrostatic interactions play a role in RNA folding analogous to hydrophobic interactions in protein folding.
1Department of Chemistry and Chemical Biology