An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labeled small peptides
- Valeriya I. Marina1,2,10,
- Medina Bidzhieva3,4,10,
- Andrey G. Tereshchenkov2,5,
- Dmitry Orekhov6,
- Vladislava E. Sagitova2,
- Nataliya V. Sumbatyan2,
- Vadim N. Tashlitsky2,5,
- Artem S. Ferberg2,
- Tinashe P. Maviza1,
- Pavel Kasatsky3,
- Olga Tolicheva3,
- Alena Paleskava3,4,
- Vladimir I. Polshakov7,
- Ilya A. Osterman1,
- Olga A. Dontsova1,2,5,8,
- Andrey L. Konevega3,4 and
- Petr V. Sergiev1,2,5,9
- 1Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Skolkovo 121205, Russia
- 2Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
- 3Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P. Konstantinov of NRC “Kurchatov Institute,” Gatchina 188300, Russia
- 4Institute of Biomedical Systems and Biotechnologies, Peter the Great St. Petersburg Polytechnic University, Saint Petersburg 195251, Russia
- 5A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia
- 6R&D Department, VIC Animal Health, Severny, Belgorod Region 308519, Russia
- 7Faculty of Fundamental Medicine, Lomonosov Moscow State University Moscow, Moscow 119991, Russia
- 8Department of Functioning of Living Systems, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia
- 9Institute of Functional Genomics, Lomonosov Moscow State University, Moscow 119991, Russia
- Corresponding authors: petya{at}genebee.msu.ru, konevega_al{at}pnpi.nrcki.ru
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↵10 These authors contributed equally to this work.
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Handling editor: Eric Westhof
Abstract
Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1–7 amino acids, BODIPY-labeled peptides, can be monitored using urea–polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.079766.123.
- Received July 5, 2023.
- Accepted November 22, 2023.
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