Used activity assays bulk fret and single molecule

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used activity assays, bulk FRET and single-molecule FRET (smFRET) to understand how different metal ions promote folding and self-cleavage of the Oryza sativa twister ribozyme. Although most ribozymes require additional Mg 2+ for catalysis, twister inverts this expectation, requiring 20–30 times less Mg 2+ to self-cleave than to fold. Transition metals such as Co 2+ , Ni 2+ and Zn 2+ activate twister more efficiently than Mg 2+ ions. Although twister is fully active in 0.5 mM MgCl 2 , smFRET experiments showed that the ribozyme visits the folded state infrequently under these conditions. Comparison of folding and self-cleavage rates indicates that most folding events lead to catalysis, which correlates with metal bond strength. Thus, the robust activity of twister reports on transient metal ion binding under physiological conditions. © 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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1110 NATURE CHEMICAL BIOLOGY | VOL 13 | OCTOBER 2017 | ARTICLE NATURE CHEMICAL BIOLOGY DOI: 10.1038/NCHEMBIO.2459 This 1-min interval corresponds to the end of the fast phase of self-cleavage, which is the phase that is most sensitive to MgCl 2 concentration. The fraction of RNA cleaved at different MgCl 2 con- centrations is well fit by a two-state cooperative folding equilibrium with a midpoint of 135 ( ± 12) μ M MgCl 2 that saturates around 2 mM MgCl 2 ( Fig. 1f ). Thus, twister ribozyme is fully active in physiologi- cal Mg 2+ levels, which are reported to be 1–5 mM in bacteria 16,17 and 5 mM in plant phloem 18 . We also determined the MgCl 2 require- ment for self-cleavage of the unimolecular form of the Osa twister ribozyme, which has a short P3 stem ( Fig. 1a ). Although 40% of the RNA was cleaved during sample preparation ( Fig. 1g ), we were able to measure the Mg 2+ dependence of self-cleavage for the remaining precursor RNA. The unimolecular form of the twister ribozyme was even more reactive than the bimolecular form, with a midpoint of 65 ( ± 6) μ M MgCl 2 ( Fig. 1h ). Folding of twister monitored by RNase footprinting To test whether the level of twister self-cleavage correlates with the extent of folding, we compared the folding midpoints of the bimo- lecular and unimolecular twister ribozymes at 20 °C using RNase footprinting. G21 and G35 are protected from cleavage by RNase T1 when the two pseudoknots become paired ( Supplementary Figs. 2 and 3 ). Unexpectedly, both forms of the ribozyme required higher Mg 2+ concentrations to fold than to self-cleave ( Fig. 1f , h ). The midpoint for protection of G21 in the bimolecular twister with increasing MgCl 2 concentration was 2.5 mM MgCl 2 ( Fig. 1f ), 20 times higher than the midpoint for catalysis (0.135 mM MgCl 2 ). For the unimolecular ribozyme, the midpoint for protection of G21 was ~1.8 mM ( Fig. 1h ), 27 times higher than the midpoint for catalysis (0.065 mM MgCl 2 ).
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  • Spring '13
  • Nelson
  • RNA, Wind, Ribozyme, Hammerhead Ribozyme, Osa twister

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