| dc.description.abstract |
<p>Bulges are ubiquitous building blocks of the three-dimensional structure of RNA.
They help define the global structure of helices and points of flexibility allowing
for functionally important dynamics, such as binding of proteins, ligands and small
molecules to occur. This thesis utilizes a battery of nuclear magnetic resonance (NMR)
methods and a model system of RNA bulge motifs, the transactivation response element
(TAR) RNA from the human immunodeficiency virus type 1 (HIV-1), to characterize the
dynamic energy landscape of bulges. Specifically investigating how it varies with
bulge length, divalent cations, and in the presence of epi-transcriptomic modifications.</p><p> Deleting
a single bulge residue (C24) from trinucleotide HIV-1 TAR bulge shifts a pre-existing
equilibrium from the unstacked to a stacked conformation in which the bulge residues
flip out of the helix and are highly flexible at the picosecond-to-nanosecond timescale.
However, the mutation minimally impacts microsecond-to-millisecond conformational
exchange directed towards two low-populated and short-lived excited conformational
states that form through a reshuffling of bases pairs throughout TAR. The mutant does,
however, adopt a slightly different excited conformational state on the millisecond
timescale. Therefore, minor changes in bulge topology preserve motional modes occurring
over the picosecond-to-millisecond timescales but alter the relative populations of
the sampled states or cause subtle changes in their conformational features.</p><p> The
impact of more broadly varying the length of the TAR poly-pyrimidine bulge (n = 1,
2, 3, 4 and 7) on inter-helical dynamics has been studied across a range of Mg2+ concentrations.
In the absence of Mg2+ (25 mM monovalent salt), n 3 bulges adopt predominantly unstacked
conformations (stacked population <15%) whereas 1-bulge and 2-bulge motifs adopt predominantly
stacked conformations (stacked population >85%). The 2-bulge motif is biased toward
linear conformations and increasing the bulge length leads to broader inter-helical
distributions and structures that are on average more kinked. In the presence of 3
mM Mg2+, the helices predominantly coaxially stack (stacked population >75%), regardless
of bulge length, and the midpoint for the Mg2+-dependent stacking transition does
not vary substantially (within 3-fold) with bulge length. In the absence of Mg2+,
the difference between the free energy of inter-helical coaxial stacking across the
bulge variants is estimated to be ~2.9 kcal/mol, based on an NMR chemical shift mapping
approach, with stacking being more energetically disfavored for the longer bulges.
This difference decreases to ~0.4 kcal/mol in the presence of 3 mM Mg2+. It is proposed
that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state
with increasing bulge length thus increasing the number of co-axial conformations
that can be sampled. </p><p> N6-Methyladenosine (m6A) and N1-Methylpurine (m1A and
m1G) xx or just refer to m1G?xx are post-transcriptional RNA modifications that are
proposed to influence RNA function through mechanisms that can involve modulation
of RNA structure. m6A is thought to modulate RNA structure by destabilizing base pairing.
Here, it is shown that m6A can stabilize A-U base pairing and overall RNA structure
when placed within the context of a bulge motif. m1A has also been shown to potently
destabilize RNA duplexes due to their inability to favorably accommodate Hoogsteen
base pairing. It is shown that such Hoogsteen base pairs can form in RNA when placed
in the context of a bulge motif. </p><p> Taken together, the studies show that the
dynamic energy landscape of polypyridine bulges is highly robust with respect to changes
in bulge length allowing for gradual variations in the population and energetics of
common conformations. Mg2+ plays an important role in smoothening these variations
most likely by diminishing electrostatic contributions that could vary significantly
across bulges of different length. The results also show that the structural impact
of epi-transcriptomic modifications can be greatly altered relative to duplex RNA
when targeting bulge motifs.</p>
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