Biology subjects

Livenskyi, A.

Publications and source records attributed to Livenskyi, A..

5 recordsLinked to original sources

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

Specificity and Mechanism of tRNA cleavage by the AriB Toprim nuclease of the PARIS bacterial immune system

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB effector of the PARIS defence system is a Toprim nuclease previously shown to target the E. coli tRNALys(UUU), but not a tRNALys(UUU) variant encoded by bacteriophage T5. We demonstrate here that the T5-tRNALys(UUU) is required but not sufficient to bypass PARIS immunity. Combining tRNA-sequencing, genetics, phage infection and in vitro biochemical data, we reveal that the E. coli tRNAThr(UGU), is another prime target of AriB and tRNAAsn(GUU) represents a secondary, yet biologically relevant, target of the PARIS effector. Activated AriB protein cleaves these targets in vitro, and the cleavage reaction is not dependent on the presence of specific tRNA modifications. We show that the overexpression of phage T5 tRNALys(UUU), tRNAThr(UGU) and tRNAAsn(GUU) variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose a model for tRNA recognition by the AriB dimer and provide molecular details of its nuclease activity and specificity.

microbiology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus

Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate dependent response to provide the immunity.

microbiology↗

bioRxiv2020

Histidine-Triad Hydrolases Provide Resistance to Peptide-Nucleotide Antibiotics

The Escherichia coli microcin C (McC) and related compounds are potent Trojan-horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by non-specific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This non-hydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside of the producing cells, special mechanisms evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by stand-alone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive. IMPORTANCEUncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine triad hydrolases provide resistance to microcin C - a potent inhibitor of bacterial protein synthesis.

biochemistry↗