Exploration of a maybe not so rugged RNA folding landscape

Cassidy E. Crook, Jrg Schlatterer and Michael Brenowitz Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Abstract
The folding of large non-coding RNAs to their biological active three-dimensional structures often proceeds through rugged folding landscapes dominated by parallel pathways and wellpopulated intermediates. Global compaction of a polyribonucleotide chain induced by non-specific cation-mediated backbone charge neutralization facilitates rapid chain collapse, followed by the organization of contiguous coaxial helices as well as tertiary motifs between disparate regions of a given molecule. We present studies of the folding kinetics in the phage Twort group I self-splicing ribozyme. The folding of the Twort ribozyme was recently described by time-resolved hydroxyl radical footprinting and catalytic activity determination in comparison with the group I ribozymes from Tetrahymena and Azoarcus (Mitra et al 2011). The folding of the Twort ribozyme is distinguished from the other two by its condensed folding time-scale and dominant folding pathways populated by short-lived minimally ordered intermediates. Furthermore, a recent crystal structure identifying Twort bound to the Tyr-tRNA synthase, CYT18, which is thought to function as a chaperone in folding, underscores growing interest in Twort as a model in RNA folding and the need for a more detailed understanding of its energy landscape (Paukstelis et al 2008). Here, we present studies of Twort ribozyme folding conducted as a function of temperature to discern the energetic barriers for discrete transitions in the folding pathway.
Fe-EDTA

Mixing RNA Mg2+ + H2O2

Fig 1. A three syringe quench flow mixer is used to interrogate folding with msec time resolution. Drive syringes (red) mix the 32P-RNA sample with solution containing Mg2+ (to initiate folding) and H2O2 (the substrate for the Fenton reaction) into the aging loop (green). The RNA folding reaction proceeds for a programmed period at which time a second push mixes the sample with Fe(II)-EDTA generating hydroxyl radical. The footprinting reaction is quench by expelling the solution into ethanol.

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Fig 5. Preliminary Erying analysis of the rates determined for the fast, medium, and slow clusters of Twort. The solid line is the fit of the rate constants to the Eyring expression: ln(kcat/T)=lnkB/h+(S/R)-(H/RT) Unfortunately the calculated parameters are not significant. Revalidation of the scaling of the individual time-progress curves to the transition endpoints, double exponential analysis and perhaps interrogation of additional temperatures may be required to resolve the thermodynamic parameters of this folding reaction.

Discussion
The folding of the Twort and Tetrahymena group I ribozymes are both characterized by a folding hierarchy that is grouped into three distinct kinetic clusters. While thermodynamic parameters can not yet be resolved, the time span over which the Twort ribozyme folds decreases with increasing temperature suggesting that folding intermediates are more highly populated at lower temperature. It is not yet clear whether the folding mechanism for Twort is invariant with temperature as observed for Tetrahymena (data not shown). Since the peripheral contacts of Twort are weaker than those of Tetrahymena, the expectation is that the folding barriers to Twort folding will be lower. The time dependence of the three kinetic clusters observed for folding of the Tetrahymena ribozyme show distinct thermodynamic signatures. 1) The barrier to folding the P4-P6 domain (fast cluster) is less than that to folding the catalytic core (slow cluster). Both the enthalpy and entropy resolved for the catalytic core are greater than those of the P4-P6 domain; 2) The progress curves cluster for the periphery (intermediate cluster) resolves into two distinct kinetic phases. Interestingly, the thermodynamic parameters resolved from the first phase match P4-P6 closely while those of the second phase recapitulate the catalytic core. The P4-P6 domain appears to serve as a scaffold for the initial structuring of the periphery while the additional peripheral contacts that constitute the final form structure along with the catalytic core.

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J. Schlatterer, J. Martin, A. Laederach, M. Brenowitz, manuscript in preparation

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Fig 6. Secondary structure of the Tetrahymena Sca-L21 RNA. Nucleotides in green, red, and blue reflect protections reporting solvent accessibility changes of the RNA backbone during Mg2+ induced structure formation.

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Fig 2. Quantitation and analysis of a time-resolved hydroxyl radical footprinting experiment following the Mg 2+-mediated folding of the Twort ribozyme.

Methods
Time-resolved hydroxyl radical footprinting was conducted to monitor the formation of tertiary contacts from 2.5 msec to 60 sec (Shcherbakova et al., 2006). The resolved time-progress curves were clustered through by k-means clustering. The optimal number of clusters was determined by the Gap Statistic. The cluster centroids were fit to a single exponential and the resultant rate constants analysed against the Eyring equation to obtain the activation enthalpies and entropies.

Twort

Fig 7. Summary of temperature dependent cluster affiliation of 23 local probes. Red, green, and blue rectangles correspond to the association with the fast, intermediate and slow folding cluster, respectively.

References
Golden, B., Kim, H., Chase, E. (2005) Nature. 12:82-89. Laederach, A., Shcherbakova, I., Liang, M.P., Brenowitz, M., Altman, RB. (2006) J. Mol Biol. 358:1179-90. Mitra, S., Laederach, A., Golden, BL., Altman, RB., Brenowitz, M. (2011) RNA. 17: 1-15. Silverman, SK., Deras, M.L., Woodson, SA., Scaringe, SA., Cech, TR. (2000) Biochem. 39: 12465-75. Shlatterer, J., Martin, J., Laederach, A., Brenowitz, M. (in preparation). Mapping the competing barriers that define RNA folding. Tibshirani, R. Walther, G., Hastie, T. (2001) J. Royal Stat Soc. 63:411-23. Paukstelis, PJ, Chen, JH., Chase, E., Lambowitz, AM., Golden, B. (2008) Nature. 451:94-98. Shcherbakova, I., Mitra, S., Beer, R., Brenowitz, M. (2006) Nucleic Acids Research. 34: 1-9.

Fig 3. The tertiary and secondary structures of the Twort ribozyme. The 3-D rendition of the molecule is displayed using a ribbon diagram from PDB entry 1Y0Q. Protected regions of the RNA are displayed in blue.
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Fig 4. The clustered time-progress curves mapping the change in solvent accessible surface for individual or groups of nucleotides distributed throughout the Twort ribozyme. The progress curves have been fit to a single exponential in this preliminary analysis.

Fig 8. Time dependence of the individual clusters. (A) The left and right panel show the simulated time progression curves derived from the global fit of each cluster to the Erying equation. The time progressions of the clusters are shown at 25 C (solid line), 40 C (dashed line), and 51 C (dotted line). (B) The kinetic rate constants of the fast (left), slow (right), and intermediate (middle) clusters are presented in Eyring plots.

Contact author: Cassidy.Crook@gmail.com