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Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes from Rabbit Reticulocyte Lysate Suitable for
Javier Rol-Moreno1, Lauriane Kuhn2, Stefano Marzi1
1Architecture et Réactivité de l'ARN - CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France.
Methods in Molecular Biology (Clifton, N.J.)
|February 2, 2020
Summary
We developed Grad-cryo-EM to study protein synthesis initiation complexes (ICs) in near-native conditions. This method uses cryo-electron microscopy and mass spectrometry to analyze translation intermediates from cell extracts.
Area of Science:
- Structural Biology
- Molecular Biology
- Biochemistry
Background:
- Single-particle cryo-electron microscopy (cryo-EM) is crucial for studying ribosomal complexes.
- Advancements in cryo-EM enable atomic resolution structural analysis of the translation machinery.
- Current methods often rely on in vitro assembly, limiting analysis to well-characterized complexes.
Purpose of the Study:
- To describe a novel method for isolating and analyzing translation initiation complexes (ICs).
- To investigate ICs assembled on natural mRNA directly from cell extracts.
- To enable structural and compositional analysis of translation intermediates in near-native conditions.
Main Methods:
- Isolation of ICs using sucrose density gradient centrifugation from rabbit reticulocyte lysate (RRL).
- Application of cryo-electron microscopy (cryo-EM) for structural determination.
- Utilizing mass spectrometry for compositional analysis of isolated complexes.
Main Results:
- The Grad-cryo-EM approach successfully isolates and characterizes intermediate ribosomal complexes.
- The method allows for near-native preparation of complexes for cryo-EM and mass spectrometry.
- Enables detailed investigation of the dynamic protein synthesis initiation process.
Conclusions:
- Grad-cryo-EM is a powerful technique for studying translation initiation.
- This method can be applied to various mRNAs, including IRES-containing ones.
- The approach is adaptable to different cell extracts, broadening its applicability.
