Abstract
The study of biologically relevant native RNA structures is important to understand their cellular function(s). Native gel electrophoresis provides information about such native structures in solution as a function of experimental conditions. The application of native gel electrophoresis in a comparative manner allows to obtain precise information on relative angles subtended between given pair of stems in an RNA molecule. By adapting this approach, it is possible to obtain very specific structural information such as the amplitude of dihedral angles and helical rotation. As an example, we will describe how native gel electrophoresis can be used to study the folding of the S-adenosylmethionine (SAM) sensing riboswitch.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lilley DM (1998) Folding of branched RNA species. Biopolymers 48:101–112
Lescoute A, Westhof E (2006) Topology of three-way junctions in folded RNAs. RNA 12:83–93
Westhof E, Fritsch V (2000) RNA folding: beyond Watson-Crick pairs. Structure 8:R55–R65
Ban N, Nissen P, Hansen J et al (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920
Wimberly BT, Brodersen DE, Clemons WM Jr et al (2000) Structure of the 30S ribosomal subunit. Nature 407:327–339
Serganov A, Patel DJ (2012) Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Annu Rev Biophys 41:343–370
Lilley DM (2009) The structure and folding of branched RNA analyzed by fluorescence resonance energy transfer. Methods Enzymol 469:159–187
Lilley DM (2004) Analysis of global conformational transitions in ribozymes. Methods Mol Biol 252:77–108
Lilley DM (2000) Structures of helical junctions in nucleic acids. Q Rev Biophys 33:109–159
Lilley DM (2000) Analysis of global conformation of branched RNA species using electrophoresis and fluorescence. Methods Enzymol 317:368–393
Lumpkin OJ (1982) Mobility of DNA in gel electrophoresis. Biopolymers 21:2315–2316
Duckett DR, Murchie AI, Diekmann S et al (1988) The structure of the Holliday junction, and its resolution. Cell 55:79–89
Ortiz-Lombardia M, Gonzalez A, Eritja R et al (1999) Crystal structure of a DNA Holliday junction. Nat Struct Biol 6:913–917
Eichman BF, Vargason JM, Mooers BH et al (2000) The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions. Proc Natl Acad Sci USA 97:3971–3976
Heppell B, Blouin S, Dussault AM et al (2011) Molecular insights into the ligand-controlled organization of the SAM-I riboswitch. Nat Chem Biol 7:384–392
Lafontaine DA, Norman DG, Lilley DM (2002) The global structure of the VS ribozyme. EMBO J 21:2461–2471
McDaniel BA, Grundy FJ, Henkin TM (2005) A tertiary structural element in S box leader RNAs is required for S-adenosylmethionine-directed transcription termination. Mol Microbiol 57:1008–1021
Heppell B, Lafontaine DA (2008) Folding of the SAM aptamer is determined by the formation of a K-turn-dependent pseudoknot. Biochemistry 47:1490–1499
Pleiss JA, Derrick ML, Uhlenbeck OC (1998) T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates. RNA 4:1313–1317
Montange RK, Batey RT (2006) Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441:1172–1175
Acknowledgements
This work was supported by the National Sciences and Engineering Research Council of Canada (NSERC). DAL is a Canadian Institutes of Health Research (CIHR) New Investigator Scholar.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Eschbach, S.H., Lafontaine, D.A. (2014). RNA Conformational Changes Analyzed by Comparative Gel Electrophoresis. In: Waldsich, C. (eds) RNA Folding. Methods in Molecular Biology, vol 1086. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-667-2_15
Download citation
DOI: https://doi.org/10.1007/978-1-62703-667-2_15
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-666-5
Online ISBN: 978-1-62703-667-2
eBook Packages: Springer Protocols