Abstract
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.
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References
Guinier A, Fournet G (1955) Small-angle scattering of X-ray. Wiley, New York
Feigin LA, Svergun DI (1987) Structure analysis by small-angle X-ray and Neutron scattering. Plenum, New York
Pollack L (2011) Time resolved SAXS and RNA folding. Biopolymers 95:543–549
Baird N, Westhof E, Qin H, Pan T, Sosnick T (2005) Structure of a folding intermediate reveals the interplay between core and peripheral elements in RNA Folding. J Mol Biol 352:712–722
Russell R, Millett IS, Doniach S, Herschlag D (2000) Small angle X-ray scattering reveals a compact intermediate in RNA folding. Nat Struct Biol 7:367–370
Moghaddam S, Caliskan G, Chauhan S, Hyeon C, Briber RM, Thirumalai D, Woodson SA (2009) Metal ion dependence of cooperative collapse transitions in RNA. J Mol Biol 393:753–764
Baird NJ, Kulshina N, Ferré-D'Amaré AR (2010) Riboswitch function: flipping the switch or tuning the dimmer? RNA Biol 7:328–332
Baird NJ, Ferré-D'Amaré AR (2010) Idiosyncratically tuned switching behavior of riboswitch aptamer domains revealed by comparative small-angle X-ray scattering analysis. RNA 16:598–609
Stoddard CD, Montange RK, Hennelly SP, Rambo RP, Sanbonmatsu KY, Batey RT (2010) Free state conformational sampling of the SAM-I riboswitch aptamer domain. Structure 18:787–797
Ali M, Lipfert J, Seifert S, Herschlag D, Doniach S (2010) The ligand-free state of the TPP riboswitch: a partially folded RNA structure. J Mol Biol 396:153–165
Lipfert J, Das R, Chu VB, Kudaravalli M, Boyd N, Herschlag D, Doniach S (2007) Structural transitions and thermodynamics of a glycine-dependent riboswitch from Vibrio cholerae. J Mol Biol 365:1393–1406
Lipfert J, Sim AY, Herschlag D, Doniach S (2010) Dissecting electrostatic screening, specific ion binding, and ligand binding in an energetic model for glycine riboswitch folding. RNA 16:708–719
Lipfert J, Chu VB, Bai Y, Herschlag D, Doniach S (2007) Low-resolution models for nucleic acids from small-angle X-ray scattering with applications to electrostatic modeling. J Appl Crystallogr 40:229–234
Wood S, Ferre-D'Amare AR, Rueda D (2012) Allosteric tertiary interactions preorganize the c-di-GMP riboswitch and accelerate ligand binding. ACS Chem Biol 7:759–770
Batey RT, Kieft JS (2007) Improved native affinity purification of RNA. RNA 13:1384–1389
Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798
Montange RK, Mondragón E, Van Tyne D, Garst AD, Ceres P, Batey RT (2010) Discrimination between closely related cellular metabolites by the SAM-I riboswitch. J Mol Biol 396:761–772
Baird NJ, Zhang J, Hamma T, Ferré-D'Amaré AR (2012) YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops. RNA 18:759–770
Svergun DI, Bargerato C, Koch MHJ (1995) CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773
Garst AD, Héroux A, Rambo RP, Batey RT (2008) Crystal structure of the lysine riboswitch regulatory mRNA element. J Biol Chem 283:22347–22351
Doniach S (2001) Changes in biomolecular conformation seen by small angle X-ray scattering. Chem Rev 101:1763–1778
Kratky O, Porod G (1949) Rontgenuntersuchung geloster fadenmolekule. Recueil des travaux chimiques des pays-bas Journal of the Royal Netherlands Chemical Society 68:1106–1122
Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503
Franke D, Svergun DI (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Crystallogr 42:342–346
Kulshina N, Baird NJ, Ferré-D'amaré AR (2009) Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch. Nat Struct Mol Biol 16:1212–1217
Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr 36:860–864
Jacques DA, Trewhella J (2010) Small-angle scattering for structural biology-Expanding the frontier while avoiding the pitfalls. Protein Sci 19:642–657
Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E, Ferrin T (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612
Acknowledgments
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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Baird, N.J., Ferré-D’Amaré, A.R. (2014). Analysis of Riboswitch Structure and Ligand Binding Using Small-Angle X-ray Scattering (SAXS). In: Lafontaine, D., Dubé, A. (eds) Therapeutic Applications of Ribozymes and Riboswitches. Methods in Molecular Biology, vol 1103. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-730-3_16
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