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Single-molecule analysis of Mss116-mediated group II intron folding

Abstract

DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ−D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg2+ concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATP-dependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly.

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Figure 1: Single-molecule fluorescence detection of group II intron folding with its natural cofactor Mss116.
Figure 2: Effect of ionic strength on the folding dynamics of D135-L14 ribozyme.
Figure 3: Mss116 promotes folding of group II introns at near-physiological conditions.
Figure 4: Role of ATP in Mss116-mediated folding of group II introns.
Figure 5: Mss116-mediated group II intron folding.

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Acknowledgements

We thank R. K. Sigel and O. Fedorova for many helpful and stimulating discussions and for commenting on the manuscript, and A. Feig, K. Musier-Forsyth and R. Lamichhane for protein gifts. This work was supported by the National Institutes of Health (R01GM085116 to D.R., R01GM050313 to A.M.P.) and the National Science Foundation (MCB-0747285 to D.R.). A.M.P. is an HHMI investigator.

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D.R. and A.M.P. conceived and designed the experiments. K.S.K. performed and analysed the experiments with help from A.S. All authors wrote the manuscript.

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Correspondence to Anna Marie Pyle or David Rueda.

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The authors declare no competing financial interests.

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Karunatilaka, K., Solem, A., Pyle, A. et al. Single-molecule analysis of Mss116-mediated group II intron folding. Nature 467, 935–939 (2010). https://doi.org/10.1038/nature09422

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