Abstract
Translation initiation, in both eukaryotes and bacteria, requires essential elements such as mRNA, ribosome , initiator tRNA, and a set of initiation factors. For each domain of life, canonical mechanisms and signals are observed to initiate protein synthesis. However, other initiation mechanism can be used, especially in viral mRNAs. Some viruses hijack cellular machinery to translate some of their mRNAs through a noncanonical initiation pathway using internal ribosome entry site (IRES), a highly structured RNAs which can directly recruit the ribosome with a restricted set of initiation factors, and in some cases even without cap and initiator tRNA. In this chapter, we describe the use of biosensors relying on electro-switchable nanolevers using the switchSENSE® technology, to investigate kinetics of the intergenic (IGR) IRES of the cricket paralysis virus (CrPV) binding to 80S yeast ribosome . This study provides a proof of concept for the application of this method on large complexes.
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References
Balvay L, Soto Rifo R, Ricci EP et al (2009) Structural and functional diversity of viral IRESes. Biochim Biophys Acta 1789(9-10):542–557. https://doi.org/10.1016/j.bbagrm.2009.07.005
Kieft JS (2008) Viral IRES RNA structures and ribosome interactions. Trends Biochem Sci 33(6):274–283. https://doi.org/10.1016/j.tibs.2008.04.007
Thompson SR (2012) Tricks an IRES uses to enslave ribosomes. Trends Microbiol 20(11):558–566. https://doi.org/10.1016/j.tim.2012.08.002
Filbin ME, Kieft JS (2009) Toward a structural understanding of IRES RNA function. Curr Opin Struct Biol 19(3):267–276. https://doi.org/10.1016/j.sbi.2009.03.005
Johnson AG, Grosely R, Petrov AN, Puglisi JD (2017) Dynamics of IRES-mediated translation. Philos Trans R Soc Lond Ser B Biol Sci 372(1716). https://doi.org/10.1098/rstb.2016.0177rstb.2016.0177
Bugaud O, Barbier N, Chommy H et al (2017) Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy. RNA 23(11):1626–1635. https://doi.org/10.1261/rna.061523.117rna.061523
Petrov A, Grosely R, Chen J et al (2016) Multiple parallel pathways of translation initiation on the CrPV IRES. Mol Cell 62(1):92–103. https://doi.org/10.1016/j.molcel.2016.03.020
Muhs M, Hilal T, Mielke T et al (2015) Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES. Mol Cell 57(3):422–432. https://doi.org/10.1016/j.molcel.2014.12.016
Murray J, Savva CG, Shin BS et al (2016) Structural characterization of ribosome recruitment and translocation by type IV IRES. Elife 5:e13567. https://doi.org/10.7554/eLife.13567
Kerr CH, Ma ZW, Jang CJ et al (2016) Molecular analysis of the factorless internal ribosome entry site in cricket paralysis virus infection. Sci Rep 6:37319. https://doi.org/10.1038/srep37319
Pestova TV, Lomakin IB, Hellen CU (2004) Position of the CrPV IRES on the 40S subunit and factor dependence of IRES/80S ribosome assembly. EMBO Rep 5(9):906–913. https://doi.org/10.1038/sj.embor.7400240
Knezevic J, Langer A, Hampel PA et al (2012) Quantitation of affinity, avidity, and binding kinetics of protein analytes with a dynamically switchable biosurface. J Am Chem Soc 134(37):15225–15228. https://doi.org/10.1021/ja3061276
Langer A, Hampel PA, Kaiser W et al (2013) Protein analysis by time-resolved measurements with an electro-switchable DNA chip. Nat Commun 4:2099. https://doi.org/10.1038/ncomms3099
Rant U, Pringsheim E, Kaiser W et al (2009) Detection and size analysis of proteins with switchable DNA layers. Nano Lett 9(4):1290–1295. https://doi.org/10.1021/nl8026789
Kroener F, Heerwig A, Kaiser W et al (2017) Electrical actuation of a DNA origami Nanolever on an electrode. J Am Chem Soc 139(46):16510–16513. https://doi.org/10.1021/jacs.7b10862
Clery A, Sohier TJM, Welte T et al (2017) switchSENSE: A new technology to study protein-RNA interactions. Methods 118-119:137–145. https://doi.org/10.1016/j.ymeth.2017.03.004
Ben-Shem A, Garreau de Loubresse N, Melnikov S et al (2011) The structure of the eukaryotic ribosome at 3.0 a resolution. Science 334(6062):1524–1529. https://doi.org/10.1126/science.1212642
Acknowledgments
Special thanks to Claire Batisse, who provided the yeast strain for 80S purification. This strain was generously given by Yusupov lab, which was elaborated in the first place by Dinman lab. The authors would like to thank Dynamic Biosensors for the encouraging collaboration. Finally, thank you to Philippe Dumas for his constant support, Yaser Hashem, Stefano Marzi, and Angelita Simonetti for fruitful discussion.
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Schenckbecher, E., Bec, G., Sakamoto, T., Meyer, B., Ennifar, E. (2021). Biophysical Studies of the Binding of Viral RNA with the 80S Ribosome Using switchSENSE. In: Daviter, T., Johnson, C.M., McLaughlin, S.H., Williams, M.A. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 2263. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1197-5_15
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