Authors
Daria S Vinogradova, Pavel S Kasatsky, Zoya A Spiridonova, Sebastian Leyva, Ana Sanchez-Castro, Katherin Peñaranda, Victor Zegarra, Pablo Soriano, Alena Paleskava, Pohl Milon, Andrey L Konevega
Published in
International journal of molecular sciences. Volume 27. Issue 11. May 29, 2026. Epub May 29, 2026.
Abstract
In prokaryotes, translation initiation orchestrates protein synthesis through a network of dynamic interactions among the ribosome, mRNA, initiator tRNAfMet, and initiation factors (IFs). Traditional approaches that rely on radioactive labeling or surface immobilization are hindered by inherent safety risks and methodological constraints. We present a fluorescence-based analytical platform that integrates microscale thermophoresis (MST) as a unified, multiparametric toolkit for comprehensive interrogation of bacterial translation initiation at the molecular level. By systematically applying MST to a panel of fluorescently labeled components-initiator tRNAfMet, mRNAs, and initiation factors-we quantify assembly pathways and equilibria as initiation progresses from simple bimolecular interactions to higher-order, multicomponent complexes. To broaden the fluorescence toolbox for ribosomal studies, we developed a robust BODIPY-labeling protocol for 70S ribosomes and confirmed preservation of structural integrity and function by nano differential scanning fluorimetry, stopped-flow kinetic assays, and peptide-synthesis activity tests. Our microscale fluorescent system facilitates probing initiation at a variety of steps, since the role of magnesium ions and initiation factors upon 30S initiation complex formation. The same platform can be applied to investigate the effects of different compounds on translation initiation, as demonstrated for a number of antibiotics, aptamers, and antimicrobial peptides. Using this approach, we determined the antibiotic streptomycin dissociation constant for both 30S and 70S ribosomes, which proved identical at 0.3 ± 0.1 μM, and demonstrated the effect of the antimicrobial peptide rumicidin-1 on translation initiation. Offering a cost-effective and high-sensitivity alternative to conventional methods, this approach advances mechanistic understanding of prokaryotic translation and provides a versatile framework for the discovery of novel protein synthesis inhibitors.
PMID:
42278479
Bibliographic data and abstract were imported from PubMed on 15 Sep 2026.
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