11 research outputs found
Effects of osmolytes and macromolecular crowders on stable GAAA tetraloops and their preference for a CG closing base pair
Osmolytes and macromolecular crowders have the potential to influence the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in vivo. To further understand the cellular function of RNA we observed the effects of a model osmolyte, polyethylene glycol (PEG) 200, and a model macromolecular crowding agent, PEG 8000, on the GAAA tetraloop motif. GAAA tetraloops are conserved, stable tetraloops, and are critical participants in RNA tertiary structure. They also have a thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing GAAA loops was monitored using UV-Vis spectroscopy in the presence and absence of PEG 200 or PEG 8000. Both of the cosolutes tested influenced the thermodynamic preference for a CG base pair by destabilizing the loop with a CG closing base pair relative to the loop with a GC closing base pair. This result also extended to a related DNA triloop, which provides further evidence that the interactions between the loop and closing base pair are identical for the d(GCA) triloop and the GAAA tetraloop. Our results suggest that in the presence of model PEG molecules, loops with a GC closing base pair may retain some preferential interactions with the cosolutes that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes could influence how neutral cosolutes tune the stability and function of secondary structure motifs in vivo
Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle Xâray Scattering
The interferon-inducible, double-stranded (ds) RNA-activated
protein
kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key
roles in viral pathogenesis and innate immunity. Activation of PKR
is typically mediated by long dsRNA, and regulation of PKR is disfavored
by most RNA imperfections, including bulges and internal loops. Herein,
we combine isothermal titration calorimetry (ITC), electrophoretic
mobility shift assays, and small-angle X-ray scattering (SAXS) to
dissect the thermodynamic basis for the specificity of the dsRBD termed
âp20â for various RNAs and to detect any RNA conformational
changes induced upon protein binding. We monitor binding of p20 to
chimeric duplexes containing terminal RNAâDNA hybrid segments
and a central dsRNA segment, which was either unbulged (âperfectâ)
or bulged. The ITC data reveal strong binding of p20 to the perfect
duplex (<i>K</i><sub>d</sub> ⌠30 nM) and weaker
binding to the bulged duplex (<i>K</i><sub>d</sub> âŒ
2â5 ÎŒM). SAXS reconstructions and <i>p</i>(<i>r</i>) distance distribution functions further uncover that
p20 induces no significant conformational change in perfect dsRNA
but largely straightens bulged dsRNA. Together, these observations
support the dsRBDâs ability to tightly bind to only A-form
RNA and suggest that in a noninfected cell, PKR may be buffered via
weak interactions with various bulged and looped RNAs, which it may
straighten. This work suggests that PKR-regulating RNAs with complex
secondary and tertiary structures likely mimic dsRNA and/or engage
portions of PKR outside of the dsRBD