The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements

Abstract In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.


Chemicals and conditions
All reactions were performed under argon atmosphere using dry solvents purchased from Acros Organics or Merck KGaA. Reagents were purchased from Acros Organics, Merck KGaA, ChemPur, TCI, Alfa Aesar or ChemGenes and used without further purification. For flash chromatography the used silica gel was purchased from Macherey-Nagel (particle size: 40-63 µm), solvents were of technical grade. NMR spectra were recorded on Bruker DPX250, AV400 and DRX600 instruments at ambient temperature.

1.2
Synthesis of the (S)-NPE caged guanosine (G S-NPE ) phosphoramidite The synthesis of the (S)-NPE protected guanosine phosphoramidite was performed according to literature procedure (1).

NMR spectroscopy
NMR experiments were performed on Bruker NMR spectrometers with different probe heads listed in Table S 4. NMR experiments were performed with standard Bruker pulse sequences and spectra were recorded and analyzed with TopSpin 3.5pl5-7. All samples containing 10% D2O and the same buffer: 50 mM BisTris, 25 mM NaCl, pH 6.4. 1D 1 H imino proton spectra were recorded using a jump return echo pulse sequence. Thermal equilibration for all samples at each temperature was done for at least 20 minutes before the experiments were recorded.

Analysis of uncaged RNA sample
An analytical denaturing 15% PAGE (polyacrylamide) was performed to ensure that laser light, StpA-CTD or PEG-8000 do not degrade uncaged RNA. Uncaged RNAs do not show degradation, also in complex with StpA-CTD or in presence of PEG-8000. After uncaging the RNA is at the same horizontal position as unmodified RNA.

Fit of kinetics rates
The individual kinetic traces of the imino proton signals were extracted from the pseudo 2D kinetic spectra.

Kinetics under molecular Crowding conditions with PEG-8000
For these experiments at 278 K and 298 K RNA and RNA with 8 %(w/v) PEG-800 was uncaged as described previous. The kinetic traces were evaluated as described in SI 7.3. The kinetic traces for PEG-8000 and for the RNA alone, both were fitted with the corresponding equlibirum constant for the RNA ate the corresponding temperature (see SI Table S

Water exchange rates
Pseudo 2D water exchange NMR experiments were conducted and evaluated as described in Rinnenthal et al. (3).