Fast Folding of RNA Pseudoknots Initiated by Laser Temperature-Jump

Abstract

RNA pseudoknots are examples of minimal structural motifs in RNA with tertiary interactions that stabilize the structures of many ribozymes. They also play an essential role in a variety of biological functions that are modulated by their structure, stability, and dynamics. Therefore, understanding the global principles that determine the thermodynamics and folding pathways of RNA pseudoknots is an important problem in biology, both for elucidating the folding mechanisms of larger ribozymes as well as addressing issues of possible kinetic control of the biological functions of pseudoknots. We report on the folding/unfolding kinetics of a hairpin-type pseudoknot obtained with microsecond time-resolution in response to a laser temperature-jump perturbation. The kinetics are monitored using UV absorbance as well as fluorescence of extrinsically attached labels as spectroscopic probes of the transiently populated RNA conformations. We measure folding times of 1-6 ms at 37 °C, which are at least 100-fold faster than previous observations of very slow folding pseudoknots that were trapped in misfolded conformations. The measured relaxation times are remarkably similar to predictions of a computational study by Thirumalai and co-workers (Cho, S. S.; Pincus, D.L.; Thirumalai, D. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17349-17354). Thus, these studies provide the first observation of a fast-folding pseudoknot and present a benchmark against which computational models can be refined.

Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

“…together with probes such as tryptophans and fluorescent nucleotide analogs, has led to the characterization of otherwise unresolved conformational dynamics during protein folding (42)(43)(44)(45), RNA folding (46), and protein-DNA (47-49) and protein-RNA (50) interactions.…”

Ensemble simulations: folding, unfolding and misfolding of a high-efficiency frameshifting RNA pseudoknot

“…Our observation of multiple intermediates in pseudoknot folding agrees with numerous previous observations and predictions including the simple model of Ansari et al . based on laser T-jump experiments (32) and the folding pathways predicted from coarse-grained simulations by Thirumalai et al . (33).…”

“…While the M 1 through M 3 misfolds might easily be clustered or classified into a larger number of misfolded states, our analysis has emphasized macroscopically measurable structural characteristics and both M 2 and M 3 represent misfolds predicted by the statistical mechanical model of Ansari et al . (32). …”

Coarse‐Grained and Atomistic MD Simulations of RNA and DNA Folding

“…Indeed, even simple RNA hairpins and pseudoknots have complex folding kinetics. [30,[34][35][36][37] However, the similar themes imply that the same formalism and concepts from protein folding can serve as a basis for the study of nucleic acid folding mechanisms. [32,38] 3 Bottom-Up and Top-Down Approaches for Biomolecular MD Simulations MD simulations continue to be an indispensable tool for the study of biomolecular folding and assembly mechanisms.…”

“…[30] The folding times predicted by the TIS model were remarkably similar to the relaxation times measured by Ansari and coworkers in a subsequent laser temperature-jump perturbation study. [37] Inspired by interrupted multiple ion-jump single-molecule experiments, [67] Biyun et al developed novel ion-concentration induced jump TIS model MD simulations by abruptly changing the ion concentration in the DebyeÀHückel potential. [36] Very recently, Li et al performed TIS model simulations of four different tRNA molecules of similar length and tertiary structure but different sequence.…”