Chapter 15
Biophysical Studies of the Binding of Viral RNA with the 80S
Ribosome Using switchSENSE
Emma Schenckbecher, Guillaume Bec, Taiichi Sakamoto, Benoit Meyer,
and Eric Ennifar
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 electroswitchable 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.
Key words Kinetics, switchSENSE, Ribosome, RNA, Biophysics
1 Introduction
Due to its key role in the cell, translation machinery has been the
subject of intense studies, especially since access to high-resolution
structures has been facilitated by X-ray crystallography and cryoelectron microscopy studies. This includes studies focusing on the
IRES-mediated initiation such as found in viral RNAs [1–3]. A
plethora of studies investigated interactions between various viral
IRES (internal ribosomal entry site) and the ribosome [4, 5], especially through the work on the intergenic (IGR) IRES of the cricket
paralysis virus (CrPV) which is now well characterized. Numerous
approaches are available to characterize interactions between such
macromolecular complexes, whether from a kinetic [6, 7], structural [8, 9], or biochemical point of view [10, 11]. Among all new
emerging techniques, switchSENSE
® technology appears as very
innovative in the solid-support immobilization field [12–14]. By
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
341
Biophysical Studies of the Binding of Viral RNA with the 80S
Ribosome Using switchSENSE
Emma Schenckbecher, Guillaume Bec, Taiichi Sakamoto, Benoit Meyer,
and Eric Ennifar
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 electroswitchable 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.
Key words Kinetics, switchSENSE, Ribosome, RNA, Biophysics
1 Introduction
Due to its key role in the cell, translation machinery has been the
subject of intense studies, especially since access to high-resolution
structures has been facilitated by X-ray crystallography and cryoelectron microscopy studies. This includes studies focusing on the
IRES-mediated initiation such as found in viral RNAs [1–3]. A
plethora of studies investigated interactions between various viral
IRES (internal ribosomal entry site) and the ribosome [4, 5], especially through the work on the intergenic (IGR) IRES of the cricket
paralysis virus (CrPV) which is now well characterized. Numerous
approaches are available to characterize interactions between such
macromolecular complexes, whether from a kinetic [6, 7], structural [8, 9], or biochemical point of view [10, 11]. Among all new
emerging techniques, switchSENSE
® technology appears as very
innovative in the solid-support immobilization field [12–14]. By
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_15, © Springer Science+Business Media, LLC, part of Springer Nature 2021
341