Ligand-modulated folding of the full-length adenine riboswitch probed by NMR and single-molecule FRET spectroscopy

Abstract

The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.

Allosteric Mechanism of theV. vulnificusAdenine Riboswitch Resolved by Four-dimensional Chemical Mapping

“…These observations are again consistent with NMR studies which could unambiguously detect P5 and estimate relative frequencies of, e.g., P2 vs. P2B, but could not establish these helices' absolute frequencies. Sixth, newer single molecule FRET measurements detect transient formation of the L2/L3 tertiary contact prior to adenine binding (dashed lines in apoA ofFigure 6) and stabilization upon adenine binding to holo 27. These data are consistent with, and indeed were interpreted within, an MWC framework in which formation of the aptameric secondary structure occurs in concert with opening of the gene expression platform, even without adenine ligand present 27.…”

“…As a reflection of current uncertainties in the system, the same group that first proposed an non-MWC model for the add RNA has recently revived the MWC framework to interpret newer single molecule measurements on the same system. 27 Understanding the mechanism of allostery is particularly important for developing strategies for controlling riboswitches. For example, potential antibiotics that attempt to constitutively turn on expression of the add gene by 5 selectively stabilizing the aptamer secondary structure may not succeed if the MWC model is incorrect.Here, we develop an experimental approach that resolves the allosteric mechanism of the add riboswitch.…”

“…Sixth, newer single molecule FRET measurements detect transient formation of the L2/L3 tertiary contact prior to adenine binding (dashed lines in apoA ofFigure 6) and stabilization upon adenine binding to holo 27. These data are consistent with, and indeed were interpreted within, an MWC framework in which formation of the aptameric secondary structure occurs in concert with opening of the gene expression platform, even without adenine ligand present 27. Seventh, the prior detailed NMR analysis led to a compelling model of riboswitch temperature robustness that involves compensating the improving affinity of the aptamer for adenine at colder temperatures by a pre-equilibrium that favored apoB over apoA at those temperatures.…”

Statistical Mechanical Prediction of Ligand Perturbation to RNA Secondary Structure and Application to the SAM-I Riboswitch

“…The first two categories are classified as "binding-competent" conformers (48), while the third category is considered to be "binding-incompetent". RNA structures which contain a particular substructure (that is, a specific configuration within the the P1/AT competition region) were considered indistinguishable, regardless of the configuration in other parts of the molecule (i. e. helices P2-P4 and linking segments), and likewise for those containing the Aptamer_TS.…”

“…While many studies, including X-ray and NMR structural studies, have addressed riboswitch recognition, we aimed to understand how ligand binding perturbs the folding of the RNA, and, in turn, the downstream gene expression. While ligand binding to a conformational intermediate has been documented for at least one other riboswitch (48,56), the yitJ SAM-I riboswitch is unique in that binding affinity measurements are available for a significant proportion of the likely binding-competent intermediates.…”