Analysis of Riboswitch Structure and Ligand Binding Using Small-Angle X-ray Scattering (SAXS)
Small-angle X-ray scattering (SAXS) is a powerful tool for examining the global conformation of riboswitches in solution, and how this is modulated by binding of divalent cations and small molecule ligands. SAXS experiments, which typically require only minutes per sample, directly yield two quantities describing the size and shape of the RNA: the radius of gyration (R g) and the maximum linear dimension (D max). Examination of these quantities can reveal if a riboswitch undergoes cation-induced compaction. Comparison of the R g and D max values between samples containing different concentrations of ligand reveals the overall structural response of the riboswitch to ligand. The Kratky plot (a graphical representation that emphasizes the higher-resolution SAXS data) and the P(r) plot or pair-probability distribution (an indirect Fourier transform, or power spectrum of the data) can provide additional evidence of riboswitch conformational changes. Simulation methods have been developed for generating three-dimensional reconstructions consistent with the one-dimensional SAXS data. These low-resolution molecular envelopes can aid in deciphering the relative helical arrangement within the RNA.
Key wordsRiboswitch Small-angle X-ray scattering RNA folding
The authors would like to thank K. Deigan and J. Zhang for helpful comments, L. Guo (sector 18-ID BioCAT) and S. Seifert and X. Zuo (sector 12-ID, BESSRC) for assistance with SAXS data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract No. W-31-109-ENG-38. BioCAT is a National Institutes of Health-supported Research Center RR-08630. This work was supported by the intramural program of the National Heart, Lung, and Blood Institute, NIH.
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