Biomedical Engineering Reference
In-Depth Information
A subsequent survey of RNA junctions in the Protein Data Bank (PDB)
together with molecular modeling revealed that the inter-helical trajectory
observed for TAR using RDCs is a fundamental and universal dynamic
feature of two-way junctions. The specific motional trajectory arises from
simple connectivity and steric constraints that restrict the allowed orientation
of helices along specific pathways. 151,152 These constraints were placed on a
quantitative footing and shown to provide the basis for the spatially correlated
twisting motions observed between two helices in HIV-1 TAR. 152 These
topological constraints—uncovered with the aid of RDCs—provide a blue-
print for quantitatively understanding RNA inter-helical motions acrossa
variety of junctions. 153
RDC studies, many targeting the TAR model system, have provided insights
into the dependence of inter-helical motions on various parameters of interest.
For example, different small molecules bind TAR and arrest the inter-helical
motions as well as induce co-axial stacking by variable amounts that appear
dependent on the number of cationic groups in the small molecule. 71,154,155
Likewise, increasing the concentration of Mg 2+ or Na + leads to the arrest of
TAR inter-helical motions and stabilisation of a co-axially stacked conforma-
tion. 156 These studies suggest that co-axial stacking of helices is likely
unfavorable due to negative charge repulsion, which accumulates at the
structurally confined bulge, and that interactions with cationic groups and
counterions may help alleviate this unfavorable charge repulsion. Reducing the
length of the TAR bulge linker from three to two nucleotides also resulted in
the
expected
reduction
in
the
amplitude
of
inter-helical
motions
and
stabilisation of a more co-axial TAR conformation. 71
However, the dependence of inter-helical motions on bulge linker is not
always trivial. For example, Zhang and co-workers used an order tensor
analysis of RDCs to measure inter-helical motions across the five-nucleotide
bulge in the core domain of human telomerase RNA. 157 Their results revealed
surprisingly smaller amplitude inter-helical motions than those observed across
the shorter TAR trinucleotide bulge [Figure 9.5(C)]. Here, unique stacking of
the guanine within the bulge over to the far-removed strand may serve to lock
the inter-helical structure and reduce the amplitude of inter-helical motions
observed. RDC studies are also revealing that the amplitude of inter-helical
motions can depend on the sequence of WC base pairs flanking junctions. For
example, Stelzer et al. rationally re-engineered TAR to bias the dynamic
ensemble towards the ligand-bound co-axial conformation. This was
accomplished by swapping an AU base pair with a GC base pair below the
bulge, which is expected to more favorably stack with the GC base pair in the
adjacent helix. 155 By pre-stabilising the ligand-bound state, the mutant bound
argininamide with three-fold higher affinity.
By combining domain elongation RDCs with MD simulations, Frank et al.
determined an atomic resolution dynamic ensemble for the 3-nt bulge and the
2-nt bulge of HIV-1 and HIV-2 TAR RNA, respectively. 16 The authors found
that snapshots within the dynamic ensemble closely matched ligand-bound
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