ja7b01549_si_001.pdf (2.51 MB)
Pseudoknot Formation Seeds the Twister Ribozyme Cleavage Reaction Coordinate
journal contribution
posted on 2017-06-09, 11:50 authored by Nikola Vušurović, Roger B. Altman, Daniel S. Terry, Ronald Micura, Scott C. BlanchardThe twister RNA is a recently discovered
nucleolytic ribozyme that is present in both bacteria and eukarya.
While its biological role remains unclear, crystal structure analyses
and biochemical approaches have revealed critical features of its
catalytic mechanism. Here, we set out to explore dynamic aspects of
twister RNA folding along the cleavage reaction coordinate. To do
so, we have employed both bulk and single-molecule fluorescence resonance
energy transfer (FRET) methods to investigate a set of twister RNAs
with labels strategically positioned at communicating segments. The
data reveal that folding of the central pseudoknot (T1), the most
crucial structural determinant to promote cleavage, exhibits reversible
opening and closing dynamics at physiological Mg2+ concentration.
Uncoupled folding, in which T1 formation precedes structuring for
closing of stem P1, was confirmed using pre-steady-state three-color
smFRET experiments initiated by Mg2+ injection. This finding
suggests that the folding path of twister RNA requires proper orientation
of the substrate prior to T1 closure such that the U5-A6 cleavage
site becomes embraced to achieve its cleavage competent conformation.
We also find that the cleaved 3′-fragment retains its compacted
pseudoknot fold, despite the absence of the phylogenetically conserved
stem P1, rationalizing the poor turnover efficiency of the twister
ribozyme.