3 research outputs found
Does Cation Size Affect Occupancy and Electrostatic Screening of the Nucleic Acid Ion Atmosphere?
Electrostatics
are central to all aspects of nucleic acid behavior,
including their folding, condensation, and binding to other molecules,
and the energetics of these processes are profoundly influenced by
the ion atmosphere that surrounds nucleic acids. Given the highly
complex and dynamic nature of the ion atmosphere, understanding its
properties and effects will require synergy between computational
modeling and experiment. Prior computational models and experiments
suggest that cation occupancy in the ion atmosphere depends on the
size of the cation. However, the computational models have not been
independently tested, and the experimentally observed effects were
small. Here, we evaluate a computational model of ion size effects
by experimentally testing a <i>blind</i> prediction made
from that model, and we present additional experimental results that
extend our understanding of the ion atmosphere. Giambasu et al. developed
and implemented a three-dimensional reference interaction site (3D-RISM)
model for monovalent cations surrounding DNA and RNA helices, and
this model predicts that Na<sup>+</sup> would outcompete Cs<sup>+</sup> by 1.8–2.1-fold; i.e., with Cs<sup>+</sup> in 2-fold excess
of Na<sup>+</sup> the ion atmosphere would contain an equal number
of each cation (<i>Nucleic Acids Res.</i> <b>2015</b>, <i>43</i>, 8405). However, our ion counting experiments
indicate that there is no significant preference for Na<sup>+</sup> over Cs<sup>+</sup>. There is an ∼25% preferential occupancy
of Li<sup>+</sup> over larger cations in the ion atmosphere but, counter
to general expectations from existing models, no size dependence for
the other alkali metal ions. Further, we followed the folding of the
P4–P6 RNA and showed that differences in folding with different
alkali metal ions observed at high concentration arise from cation–anion
interactions and not cation size effects. Overall, our results provide
a critical test of a computational prediction, fundamental information
about ion atmosphere properties, and parameters that will aid in the
development of next-generation nucleic acid computational models
Single-Molecule Fluorescence Reveals Commonalities and Distinctions among Natural and <i>in Vitro</i>-Selected RNA Tertiary Motifs in a Multistep Folding Pathway
Decades
of study of the RNA folding problem have revealed that
diverse and complex structured RNAs are built from a common set of
recurring structural motifs, leading to the perspective that a generalizable
model of RNA folding may be developed from understanding of the folding
properties of individual structural motifs. We used single-molecule
fluorescence to dissect the kinetic and thermodynamic properties of
a set of variants of a common tertiary structural motif, the tetraloop/tetraloop-receptor
(TL/TLR). Our results revealed a multistep TL/TLR folding pathway
in which preorganization of the ubiquitous AA-platform submotif precedes
the formation of the docking transition state and tertiary A-minor
hydrogen bond interactions form after the docking transition state.
Differences in ion dependences between TL/TLR variants indicated the
occurrence of sequence-dependent conformational rearrangements prior
to and after the formation of the docking transition state. Nevertheless,
varying the junction connecting the TL/TLR produced a common kinetic
and ionic effect for all variants, suggesting that the global conformational
search and compaction electrostatics are energetically independent
from the formation of the tertiary motif contacts. We also found that <i>in vitro</i>-selected variants, despite their similar stability
at high Mg<sup>2+</sup> concentrations, are considerably less stable
than natural variants under near-physiological ionic conditions, and
the occurrence of the TL/TLR sequence variants in Nature correlates
with their thermodynamic stability in isolation. Overall, our findings
are consistent with modular but complex energetic properties of RNA
structural motifs and will aid in the eventual quantitative description
of RNA folding from its secondary and tertiary structural elements
Roles of Long-Range Tertiary Interactions in Limiting Dynamics of the <i>Tetrahymena</i> Group I Ribozyme
We determined the
effects of mutating the long-range tertiary contacts
of the <i>Tetrahymena</i> group I ribozyme on the dynamics
of its substrate helix (referred to as P1) and on catalytic activity.
Dynamics were assayed by fluorescence anisotropy of the fluorescent
base analogue, 6-methyl isoÂxanthoÂpterin, incorporated
into the P1 helix, and fluorescence anisotropy and catalytic activity
were measured for wild type and mutant ribozymes over a range of conditions.
Remarkably, catalytic activity correlated with P1 anisotropy over
5 orders of magnitude of activity, with a correlation coefficient
of 0.94. The functional and dynamic effects from simultaneous mutation
of the two long-range contacts that weaken P1 docking are cumulative
and, based on this RNA’s topology, suggest distinct underlying
origins for the mutant effects. Tests of mechanistic predictions via
single molecule FRET measurements of rate constants for P1 docking
and undocking suggest that ablation of the P14 tertiary interaction
frees P2 and thereby enhances the conformational space explored by
the undocked attached P1 helix. In contrast, mutation of the metal
core tertiary interaction disrupts the conserved core into which the
P1 helix docks. Thus, despite following a single correlation, the
two long-range tertiary contacts facilitate P1 helix docking by distinct
mechanisms. These results also demonstrate that a fluorescence anisotropy
probe incorporated into a specific helix within a larger RNA can report
on changes in local helical motions as well as differences in more
global dynamics. This ability will help uncover the physical properties
and behaviors that underlie the function of RNAs and RNA/protein complexes