Multistage Collapse of a Bacterial Ribozyme Observed by Time-Resolved Small-Angle X-ray Scattering

Abstract

Ribozymes must fold into compact, native structures to function properly in the cell. The first step in forming the RNA tertiary structure is the neutralization of the phosphate charge by cations, followed by collapse of the unfolded molecules into more compact structures. The specificity of the collapse transition determines the structures of the folding intermediates and the folding time to the native state. However, the forces that enable specific collapse in RNA are not understood. Using time-resolved SAXS, we report that upon addition of 5 mM Mg 2+ to the Azoarcus group I ribozyme, up to 80% of chains form compact structures in less than 1 millisecond. In 1 mM Mg 2+ , the collapse transition produces extended structures that slowly approach the folded state, while ≥ 1.5 mM Mg 2+ leads to an ensemble of random coils that fold with multi-stage kinetics. Increased flexibility of molecules in the intermediate ensemble correlates with a Mg 2+ -dependent increase in the fast folding population and a previously unobserved crossover in the collapse kinetics. Partial denaturation of the unfolded RNA with urea also increases the fraction of chains following the fast-folding pathway. These results demonstrate that the preferred collapse mechanism depends on the extent of Mg 2+ -dependent charge neutralization, and that non-native interactions within the unfolded ensemble contribute to the heterogeneity of the ribozyme folding pathways at the very earliest stages of tertiary structure formation.

The Azoarcus Group I Intron Ribozyme Misfolds and Is Accelerated for Refolding by ATP-dependent RNA Chaperone Proteins

“…Misfolding of the Azoarcus Ribozyme-Early work suggested that this ribozyme avoided long-lived intermediates and folded to the native state in milliseconds, but more recent experiments have demonstrated the presence of intermediates and slower folding steps for at least a fraction of the population (39,40). By tracking formation of the native state directly, our results build on these findings by showing that it is a large fraction, approximately half of the ribozyme, that reaches the native state slowly, on the time scale of an hour under our conditions.…”

“…Together, these results indicate that the intermediate is misfolded and must be at least partially unfolded to allow refolding to the native state. We also measured folding under conditions used in recent SAXS experiments (39). This prior work revealed a small but detectable phase of compaction, which occurred on the minutes time scale and presumably reflected slow native folding of a fraction of the ribozyme population.…”

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam

“…While stopped-flow techniques with SAXS have been used for quite some time for kinetic studies (Eliezer et al, 1995;Roh et al, 2010), time resolution has been limited by the deadtime of current commercial devices to about $ 0.5 ms. Various kinds of continuous-flow mixers, however, have the potential to go much faster (Kathuria et al, 2011).…”

Taming free energy landscapes with RNA chaperones

“…In several studies, more RNA partitioned into direct (fast) folding pathways when the RNA was prefolded in salt before Mg 2+ was added. [130][131][132][133] Partial denaturation of the unfolded Azoarcus ribozyme reduced the heterogeneity of the initial collapse kinetics, allowing more RNA to collapse in ≤1 ms. 134 Second, more RNA molecules can avoid misfolding if the forces driving helix assembly and collapse are lessened, because the RNA has more time to search for its most stable structure before the transition state for folding is crossed. 2,129 Moreover, when conditions barely favor the native state, a larger number of energetically favorable interactions must accumulate to push the RNA over the folding transition state.…”