An easy tool to monitor the elemental steps of in vitro translation via gel electrophoresis of fluorescently labelled small peptides
- Valeriya I. Marina1,
- Medina Bidzhieva2,
- Andrey G. Tereshchenkov3,
- Dmitry Orekhov4,
- Vladislava E. Sagitova3,
- Nataliya V. Sumbatyan3,
- Vadim N. Tashlitsky3,
- Artem S. Ferberg3,
- Tinashe P. Maviza1,
- Pavel Kasatsky2,
- Olga Tolicheva5,
- Alena Paleskava2,
- Vladimir I. Polshakov6,
- Ilya A. Osterman1,
- Olga A. Dontsova3,
- Andrey L. Konevega2 and
- Petr V Sergiev7,8
- 1 Skolkovo Institute of Science and Technology;
- 2 Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P. Konstantinov of NRC;
- 3 Department of Chemistry, Lomonosov Moscow State University;
- 4 R&D Department, VIC Animal Health;
- 5 Molec and Rad Biophys Div, Petersburg Nuclear Physics Inst named by BP Konstantinov of NRC Kurchatov;
- 6 Faculty of Fundamental Medicine, Lomonosov Moscow State University;
- 7 Lomonosov Moscow State University
- ↵* Corresponding author; email: petya{at}genebee.msu.ru
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 to 7 amino acids, BODIPY-labelled peptides, can be monitored using urea-polyacrylamide gel electrophoresis. Very short BODIPY-labelled 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.
Keywords
- Received July 5, 2023.
- Accepted November 22, 2023.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
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