A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate - PubMed (original) (raw)
A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate
Elliot Tan et al. Proc Natl Acad Sci U S A. 2003.
Abstract
The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules.
Figures
Fig. 1.
(a) The hairpin ribozyme used in FRET studies. The secondary structure comprises two internal loops in adjacent arms (A and B) of a four-way junction. Ribozyme folding brings the donor and acceptor fluorophores closer to each other, increasing FRET. The cleavage site is marked by an arrow; however, cleavage activity is prevented by substitution of a deoxyribonucleotide at the –1 position. (b) Stereo view of the crystal structure of the hairpin ribozyme (9), with the G+1–C25 interaction highlighted. The helical arms are coaxially stacked in pairs, A on D (magenta) and B on C (blue).
Fig. 4.
Heterogeneity in ribozyme folding and cleavage reaction kinetics. (a) Average duration of U vs. average duration of F for 125 ribozyme molecules showing up to 50-fold heterogeneity. (b) Average duration of UD vs. average duration ofU P for 62 4H junction molecules showing much narrower distribution than in a. (c) Analysis of substrate cleavage in a single ribozyme molecule with a shortened substrate so that the 3′ product is rapidly released. Upon addition of Mg2+, the molecule folds, resulting in increased FRET. After 180 s, the molecule exhibits frequent fluctuations in FRET characteristic of the 4H junction; we deduce that the substrate has now been cleaved and released, leading to loss of loop–loop interaction. (d) Fraction unreacted vs. time since Mg2+ addition for the 83 molecules that showed the ribozyme reaction before photobleaching, fitted to a single exponential decay. (e) Fraction not photobleached vs. time since Mg2+ addition for the 28 molecules that underwent photobleaching before cleavage could occur, fitted to a single exponential decay.
Fig. 2.
Structural transitions in single hairpin ribozyme and 4H junctions. Shown are single-molecule time records of _E_app for the hairpin ribozyme (a) and 4H junction (b). (c)_I_D and _I_A were measured from individual hairpin ribozymes in the unfolded state with 10-μs time resolution. Cross-correlation was averaged over at least 20 molecules under each condition. Negative cross-correlations indicate FRET changes due to conformational changes between UD andU P. Single exponential decay fits are shown. Data for 0.5 mM Mg2+ were obtained by using a G+1A variant, which does not fold at this Mg2+ concentration. (d)_E_app histogram of a single ribozyme in the unfolded state at 0.2 mM Mg2+ with two Gaussian fits. The proximal (≈0.43) and distal (≈0.15) states comprise 40% and 60% of the total population, respectively. (e) Cross-correlation between_I_D and _I_A for the 4H junction with single exponential decay fits. (f) The rate of 4H junction fluctuations (_k_D→P +_k_P→D) obtained from the data shown in e as a function of Mg2+ concentration. The data points at 2, 5, 10, 20, and 50 mM Mg2+ were obtained from the dwell-time analysis.
Fig. 3.
An intermediate folding state in a C25U variant hairpin ribozyme. (a and b) Examples of time records for two variant ribozyme molecules, with corresponding _E_app histogram [10-ms (a) and 9-ms (b) bin times]. b Inset shows an expanded plot (3-ms bin time) of the data segment marked by a dashed rectangle. (c and d) Dwell-time histograms ofU P during transitions between UD and F and single exponential decay fits. If theU P was not resolved within the bin time used, zero was assigned as the dwell time.
Fig. 5.
Proposed model for the stepwise folding of the hairpin ribozyme. The ribozyme undergoes folding into the active conformation in two stages, corresponding to the properties of the junction and the interactions between the loops. The structural dynamics of the junction promote rapid active site formation due to the spontaneous fluctuations between the two states of the junction, UD and U P. StateUD comprises a number of distinct conformations that cannot be distinguished on the basis of our current data. The ribozyme exploits the frequent encounters between the two loops in U P to achieve the F state. Single-molecule spectroscopy provides information on the previous and subsequent history of a given state, allowing us to conclude that the U P state is an obligatory intermediate in the formation of the ultimate F state. The rates shown are measured in the presence of 0.5 mM Mg2+ ions.
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