Summary
The gel mobility shift assay is routinely used to visualize protein—RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. We review the quantitative application of this approach to elucidate thermodynamic properties of protein—RNA complexes. Assay designs for titration, competition, and stoichiometry experiments are presented for two unrelated model complexes.
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References
Dahlberg, A. E., Dingman, C. W., and Peacock, A. C. (1969) Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels. J. Mol. Biol. 41, 139–147.
Schaup, H. W., Green, M., and Kurland, C. G. (1970) Molecular interactions of ribosomal components. I. Identification of RNA binding sites for individual 30S ribosomal proteins. Mol. Gen. Genet. 109, 193–205.
Murphy, F. L., Wang, Y. H., Griffith, J. D., and Cech, T. R. (1994) Coaxially stacked RNA helices in the catalytic center of the Tetrahymena ribozyme. Science 265, 1709–1712.
Samuels, M. E., Bopp, D., Colvin, R. A., Roscigno, R. F., Garcia-Blanco, M. A., and Schedl, P. (1994) RNA binding by Sxl proteins in vitro and in vivo. Mol. Cell. Biol. 14, 4975–4990.
Yakhnin, A. V. , Trimble, J. J., Chiaro, C. R., and Babitzke, P. (2000) Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis. J. Biol. Chem. 275, 4519–4524.
Vargason, J. M., Szittya, G., Burgyan, J., and Tanaka Hall, T. M. (2003) Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799– 811.
Batey, R. T., and Williamson, J. R. (1996) Interaction of the Bacillus stearother-mophilus ribosomal protein S15 with 16 S rRNA: I. Defining the minimal RNA site. J. Mol. Biol. 261, 536–549.
Zamore, P. D., Williamson, J. R., and Lehmann, R. (1997) The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. RNA 3, 1421–1433.
Ryder, S. P., Frater, L., Abramovitz, D. L., Goodwin, E. B., and Williamson, J. R. (2004) RNA target specificity of the STAR/GSG domain post-transcriptional regulatory protein GLD-1. Nat. Struct. Mol. Biol. 11, 20–28.
Recht, M. I., and Williamson, J. R. (2001) Central domain assembly: thermodynamics and kinetics of S6 and S18 binding to an S15-RNA complex. J. Mol. Biol. 313, 35–48.
delCardayre, S. B., and Raines, R. T. (1995) A residue to residue hydrogen bond mediates the nucleotide specificity of ribonuclease A. J. Mol. Biol. 252, 328–336.
Cilley, C. D., and Williamson, J. R. (1999) PACE analysis of RNA-peptide interactions. Methods Mol. Biol. 118, 129–141.
Gill, S., and von Hippel, P. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.
Cann, J. R. (1989) Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes. J. Biol. Chem. 264, 17032–17040.
Setzer, D. R. (1999) Measuring equilibrium and kinetic constants using gel retardation assays. Methods Mol. Biol. 118, 115–128.
Hill, A. V. (1910) The possible effects of the aggregation of the molecules of haemoglobin on its oxygen dissociation curve. J. Physiol. (London) 40, 4–7.
Marquardt, D. (1963) An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math. 11, 431–441.
Lin, S. Y., and Riggs, A. D. (1972) Lac repressor binding to non-operator DNA: detailed studies and a comparison of equilibrium and rate competition methods. J. Mol. Biol. 72, 671–690.
Weeks, K. M., and Crothers, D. M. (1992) RNA binding assays for Tat-derived peptides: implications for specificity. Biochemistry 31, 10281–10287.
Chen, T., Damaj, B. B., Herrera, C., Lasko, P., and Richard, S. (1997) Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain. Mol. Cell. Biol. 17, 5707–5718.
Acknowledgments
We thank Ivan Baxter for the alkaline hydrolysis protocol and Dana Abramovitz for generation of the GLD-1 expression construct. S.P.R. was supported by a Damon Runyon Cancer Research Foundation Fellowship (DRG-1723). M.I.R was supported by a Research Scholar grant from the American Cancer Society (PF-01-087-01-GMC). This research was supported by grants from the National Institutes of Health (NIH GM53320 and GM53757).
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Ryder, S.P., Recht, M.I., Williamson, J.R. (2008). Quantitative Analysis of Protein-RNA Interactions by Gel Mobility Shift. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_7
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DOI: https://doi.org/10.1007/978-1-60327-475-3_7
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