Biology subjects

Konevega, A. L.

Publications and source records attributed to Konevega, A. L..

8 recordsLinked to original sources

bioRxiv2026

A microscale platform for the comprehensive analysis of bacterial translation initiation

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) to investigate translation initiation at the molecular level. Employing fluorescently labeled molecules including the initiator tRNAfMet, mRNA, and Ifs, enabled a detailed characterization of initiation complex assembly as it progresses from bimolecular to higher-order multicomponent states. To expand the fluorescent toolbox for translation studies we established a novel BODIPY-labeling protocol for 70S ribosomes and confirmed their conformational integrity using nano differential scanning fluorimetry (nanoDSF). 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 M, 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.

biophysics↗

bioRxiv2025

Insights into the bottromycin A2 mechanism of action

The spread of antimicrobial resistance among pathogenic bacteria poses a threat for modern medicine, highlighting the need for the discovery and development of new potential therapeutic agents. Bottromycin A2 (BotA2) represents a promising candidate for future drug development, exhibiting activity against clinically relevant methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and mycoplasma. However, its exact mechanism of action has not been fully elucidated until now. Here, we demonstrate that BotA2 inhibits bacterial translation showing unique context specificity with regard to the mRNA coding sequence. By using high-throughput toe-printing combined with deep sequencing (toe-seq analysis), we show that BotA2 induces ribosome stalling predominantly when a glycine codon enters the A-site of the ribosome, with stalling efficiency independent of codons located in the P- and E-sites. Our biochemical and biophysical data reveal that BotA2 arrests glycine-containing ternary complexes on the ribosome thereby preventing the full accommodation of incoming Gly-tRNAGly in the peptidyl transferase center. Altogether, our findings uncover a completely novel, previously undescribed mechanism of translation inhibition based on the context-specific immobilization of ternary complexes on elongating ribosomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/671025v3_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@16ef44forg.highwire.dtl.DTLVardef@16984d0org.highwire.dtl.DTLVardef@8e7469org.highwire.dtl.DTLVardef@7cb57a_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

molecular biology↗

bioRxiv2025

Blocking IF3N delays bacterial translation initiation

The authors have withdrawn this manuscript because reviewers have suggested significant revisions which required major restructuring of the manuscript. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

biochemistry↗

bioRxiv2024

Stick of Sticks: Structural Features of the Amyloidogenic Peptide-DNA Complex

The search for peptides that can specifically bind to regulatory regions in DNA is a necessary step for creating drugs that can regulate gene expression. The study is dedicated to the peculiarities of binding of a model peptide, which carries an ionic self-complementary motif and can form amyloid-like fibrils [1], with model double-stranded DNAs. The stoichiometric ratios of the components of the complex were found using the retardation method in agarose gel. Using microscale thermophoresis, it was shown that the peptide in the amyloid-like state is capable of binding to model 45-bp double-stranded DNA, with a micromolar equilibrium dissociation constant. Using cryo-electron, transmission electron, and atomic force microscopy, the morphology of peptide-DNA complexes was studied. Using dynamic light scattering and nanoparticle tracking analysis, as well as small-angle neutron scattering, the spatial parameters of the resulting DNA-peptide complexes were characterized. Molecular dynamics simulations showed that the arginine side chains of the peptide are prone to interact with guanine nitrogenous bases. It was shown that the formation of peptide-dsDNA complexes interferes with the operation of restriction endonucleases that have guanine-cytosine pairs in the recognition center, which is consistent with the results of prediction of interaction sites obtained using computer modeling. The results of the work can be used in the development of peptides capable of interacting with functional regions of DNA, as well as in the development of new carriers for transfection of DNA constructs.

biophysics↗

bioRxiv2022

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin.

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site. HIGHLIGHTSO_LIThermorubin is a potent inhibitor of protein synthesis both in vivo and in vitro; C_LIO_LIThermorubin does not prevent the binding of P- and A-site tRNAs; C_LIO_LIThermorubin affects multiple steps of translation elongation with a major impact on binding stability of the A-site tRNA; C_LIO_LIThermorubin can act as an inhibitor of translation termination on some ORFs. C_LI

molecular biology↗

bioRxiv2022

Inside and outside of virus-like particles HBc and HBc/4M2e: a comprehensive study of the structure

The structure of the core particles of the hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) have been studied using a combination of molecular modeling and cryogenic electron microscopy (cryo-EM). It has also been shown that synthesis of particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e do not contain detectable amounts of bacterial proteins. It has been shown that a fragment of the M2e is prone to the formation of amyloid-like fibrils. However, as a part of HBc immunodominant loop M2e domains as repeats, does not exhibit a tendency to aggregation. Full-atom HBc-4M2E model with 3A resolution was obtained by molecular modeling methods based on cryo-EM data.

biophysics↗

bioRxiv2019

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

Initiation factor 3 (IF3) is an essential protein that enhances the fidelity and speed of bacterial initiation of mRNA translation. The dynamic interplay between the two independent IF3 domains, their alternative binding sites, and the mechanism that ensures translation initiation fidelity remains elusive. Here, we show that the functional positioning of IF3 domains occurs at velocities ranging over two orders of magnitude, driven by each 30S initiation ligand. IF1 and IF2 rapidly promote the accommodation of IF3 on the 30S platform with the C-terminal domain moving towards the P site. Reversion of this movement is triggered by decoding the mRNA start codon and rate limits translation initiation. Binding of the tRNA results in the concomitant accommodation of the N-terminal domain of IF3, largely dependent on the mRNA and initiator tRNA. 70S initiation complex formation promotes the closing and dissociation of IF3, recycling the factor for a new round of translation initiation. Altogether our results unveil the kinetic spectrum of IF3 conformations and highlight fundamental movements of the factor that ensure accurate translation initiation.

biophysics↗

bioRxiv2019

How the initiating ribosome copes with (p)ppGpp to translate mRNAs

During host colonization, bacteria use the alarmone (p)ppGpp to reshape its proteome by acting pleiotropically on RNA and protein synthesis. Here, we elucidate how the translation Initiation Factor 2 (IF2) senses the cellular ppGpp to GTP ratio and regulates the progression towards protein synthesis. Our results show that the affinity of GTP and the inhibitory concentration of ppGpp for 30S-bound IF2 vary depending on the programmed mRNA. Highly translated mRNAs enhanced GTP affinity for 30S complexes, resulting in fast transitions to elongation of protein synthesis. Less demanded mRNAs allowed ppGpp to compete with GTP for IF2, stalling 30S complexes until exchange of the mRNA enhances the affinity for GTP. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp, and that bacteria can exploit to modulate its proteome as a function of the nutritional shift happening during infection.

biochemistry↗