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A rugged free energy landscape separates multiple functional RNA folds throughout denaturation.

Ditzler MA, Rueda D, Mo J, Håkansson K, Walter NG - Nucleic Acids Res. (2008)

Bottom Line: This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data.Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure.Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.

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Multiple annealing conditions resulting in similar EMSA distributions. In the presence of monovalents alone (50 mM NaCl) or in the presence of divalents (12 mM MgCl2) 32P-labeled ribozyme (fully synthetic 2′-O-methyl construct used for mass spectrometry analysis) was subjected to the following annealing protocols: (1) 25°C for 20 min; (2) 70°C, 2 min, 25°C, 20 min; (3) 90°C, 2 min, 25°C 20 min; (4) 90°C, 2 min, 70°C, 2 min, 65°C, 2 min, 25°C, 20 min; (5) 90°C, 2 min, 25°C, 10 min repeated three times, then 70°C, 2 min, 25°C, 10 min repeated two times; (6) 90°C, 2 min, 55°C, 30 min, 25°C, 20 min. The distribution between T and B species was then analyzed by EMSA.
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Figure 2: Multiple annealing conditions resulting in similar EMSA distributions. In the presence of monovalents alone (50 mM NaCl) or in the presence of divalents (12 mM MgCl2) 32P-labeled ribozyme (fully synthetic 2′-O-methyl construct used for mass spectrometry analysis) was subjected to the following annealing protocols: (1) 25°C for 20 min; (2) 70°C, 2 min, 25°C, 20 min; (3) 90°C, 2 min, 25°C 20 min; (4) 90°C, 2 min, 70°C, 2 min, 65°C, 2 min, 25°C, 20 min; (5) 90°C, 2 min, 25°C, 10 min repeated three times, then 70°C, 2 min, 25°C, 10 min repeated two times; (6) 90°C, 2 min, 55°C, 30 min, 25°C, 20 min. The distribution between T and B species was then analyzed by EMSA.

Mentions: To examine the persistent folding heterogeneity of the hairpin ribozyme, we explored an ensemble EMSA to isolate molecular sub-populations based on their average hydrodynamic radii. During EMSA a catalytically inactivated two-way junction (2WJ) construct (Figure 1a) migrates in two bands of distinct interdomain docking equilibria (Figure 1d). This heterogeneous mobility is observed over a wide range of annealing protocols (Figure 2), in the presence or absence of a domain terminal fluorophore pair, and independent of whether catalysis is blocked by a synthetic 2′-O-methyl modification of the cleavage site A-1 or a ribozyme G8A mutation in fully in vitro transcribed material (Figures 1d, 2, 3a and 4c). The corresponding cleavable 2WJ ribozyme cleaves to 77% completion with biphasic rate constants of 0.08 and 0.01 min−1 (Figure 1b), which have been linked to more and less stably docking sub-populations, respectively (3,12). During EMSA the cleavable 2WJ ribozyme cleaves to >60% completion (Figure 1d), thereby demonstrating native folding during EMSA and necessitating inactivating modifications for the preservation, isolation and further characterization of the two bands. Additionally, we examined smFRET trajectories of a trans-cleaving t-2WJ construct where the substrate strand is separated from the remainder of the RzB strand, such that the products generated upon backbone cleavage rapidly dissociate, giving rise to a distinct FRET signal of the substrate-free ribozyme after catalysis (Figure 1c). We find that smFRET trajectories of these t-2WJ ribozymes with distinct undocking behaviors repeatedly show transitions to the self-cleaved, intermediate FRET state (Figure 1c). We therefore infer that: (i) multiple, catalytically active molecular species exist within both synthetic and transcribed hairpin ribozyme preparations (source independence), and (ii) our catalytically inactivating modifications allow us to separate molecular sub-populations for further study that reproduce the heterogeneous behavior of the catalytically active ribozyme.Figure 2.


A rugged free energy landscape separates multiple functional RNA folds throughout denaturation.

Ditzler MA, Rueda D, Mo J, Håkansson K, Walter NG - Nucleic Acids Res. (2008)

Multiple annealing conditions resulting in similar EMSA distributions. In the presence of monovalents alone (50 mM NaCl) or in the presence of divalents (12 mM MgCl2) 32P-labeled ribozyme (fully synthetic 2′-O-methyl construct used for mass spectrometry analysis) was subjected to the following annealing protocols: (1) 25°C for 20 min; (2) 70°C, 2 min, 25°C, 20 min; (3) 90°C, 2 min, 25°C 20 min; (4) 90°C, 2 min, 70°C, 2 min, 65°C, 2 min, 25°C, 20 min; (5) 90°C, 2 min, 25°C, 10 min repeated three times, then 70°C, 2 min, 25°C, 10 min repeated two times; (6) 90°C, 2 min, 55°C, 30 min, 25°C, 20 min. The distribution between T and B species was then analyzed by EMSA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2602785&req=5

Figure 2: Multiple annealing conditions resulting in similar EMSA distributions. In the presence of monovalents alone (50 mM NaCl) or in the presence of divalents (12 mM MgCl2) 32P-labeled ribozyme (fully synthetic 2′-O-methyl construct used for mass spectrometry analysis) was subjected to the following annealing protocols: (1) 25°C for 20 min; (2) 70°C, 2 min, 25°C, 20 min; (3) 90°C, 2 min, 25°C 20 min; (4) 90°C, 2 min, 70°C, 2 min, 65°C, 2 min, 25°C, 20 min; (5) 90°C, 2 min, 25°C, 10 min repeated three times, then 70°C, 2 min, 25°C, 10 min repeated two times; (6) 90°C, 2 min, 55°C, 30 min, 25°C, 20 min. The distribution between T and B species was then analyzed by EMSA.
Mentions: To examine the persistent folding heterogeneity of the hairpin ribozyme, we explored an ensemble EMSA to isolate molecular sub-populations based on their average hydrodynamic radii. During EMSA a catalytically inactivated two-way junction (2WJ) construct (Figure 1a) migrates in two bands of distinct interdomain docking equilibria (Figure 1d). This heterogeneous mobility is observed over a wide range of annealing protocols (Figure 2), in the presence or absence of a domain terminal fluorophore pair, and independent of whether catalysis is blocked by a synthetic 2′-O-methyl modification of the cleavage site A-1 or a ribozyme G8A mutation in fully in vitro transcribed material (Figures 1d, 2, 3a and 4c). The corresponding cleavable 2WJ ribozyme cleaves to 77% completion with biphasic rate constants of 0.08 and 0.01 min−1 (Figure 1b), which have been linked to more and less stably docking sub-populations, respectively (3,12). During EMSA the cleavable 2WJ ribozyme cleaves to >60% completion (Figure 1d), thereby demonstrating native folding during EMSA and necessitating inactivating modifications for the preservation, isolation and further characterization of the two bands. Additionally, we examined smFRET trajectories of a trans-cleaving t-2WJ construct where the substrate strand is separated from the remainder of the RzB strand, such that the products generated upon backbone cleavage rapidly dissociate, giving rise to a distinct FRET signal of the substrate-free ribozyme after catalysis (Figure 1c). We find that smFRET trajectories of these t-2WJ ribozymes with distinct undocking behaviors repeatedly show transitions to the self-cleaved, intermediate FRET state (Figure 1c). We therefore infer that: (i) multiple, catalytically active molecular species exist within both synthetic and transcribed hairpin ribozyme preparations (source independence), and (ii) our catalytically inactivating modifications allow us to separate molecular sub-populations for further study that reproduce the heterogeneous behavior of the catalytically active ribozyme.Figure 2.

Bottom Line: This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data.Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure.Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.

Show MeSH
Related in: MedlinePlus