Biology subjects

Klepacki, D.

Publications and source records attributed to Klepacki, D..

6 recordsLinked to original sources

bioRxiv2026

Structural modification of oxazolidinone antibiotics alters nascent peptide stalling preference and peptide trajectory through the ribosome

The oxazolidinone antibiotic linezolid binds to the peptidyl transferase center of the ribosome, where it inhibits a subset of peptide bond formation events. This context-specificity of translation inhibition is dictated by the nature of the amino acid at the penultimate position of the nascent peptide. It remains unknown whether this is a general feature of oxazolidinones and whether it can be modulated by their structural alterations. Here, we show that the oxazolidinone tedizolid also inhibits translation in a context-specific manner, but with dramatically altered selectivity, favoring Ile, His, and Gln as the penultimate residues. Delpazolid, which shares the C5 hydroxymethyl moiety with tedizolid, shows a similar preference. Structural analysis of the ribosome with tedizolid and a stalled nascent peptide showed a compacted, helical conformation of the nascent chain induced by the drug. Our findings reveal that stalling preferences of oxazolidinones can be modulated by structural modifications within this antibiotic class.

biochemistry↗

bioRxiv2025

Sequence-specific trapping of EF-Tu/glycyl-tRNA complex on the ribosome by bottromycin

The development of antibiotics with novel mechanisms of action is essential to address the growing threat of antimicrobial resistance. Protein synthesis-inhibiting antibiotic bottromycin (BOT), a ribosomally synthesized and posttranslationally modified peptide (RiPP), has long been known for its potent activity against Gram-positive bacteria but was largely neglected due in part to the lack of understanding of its mechanism of action. Here we uncover the unprecedented mode translation inhibition strategy employed by BOT. Using biochemical, microbiological, genetic, and structural approaches, we show that BOT acts by selectively trapping elongation factor-Tu (EF-Tu) in complex with glycyl-tRNA on the ribosome. BOT binds at the interface between EF-Tu and the CCA-end of Gly-tRNA, stabilizing the EF-Tu/Gly-tRNA complex in a pre-accommodated A/T-state on the ribosome, and specifically arresting translation at glycine codons. This mode of action is mechanistically distinct from that of other EF-Tu-targeting antibiotics, which act in a tRNA-agnostic fashion. Point mutations in EF-Tu confer high-level resistance to BOT, confirming EF-Tu as the direct and essential target of the drug. Our findings establish BOT as a founding member of a new class of antibiotics that stall the ribosome at defined mRNA sites by trapping a specific elongation factor-tRNA complex.

molecular biology↗

bioRxiv2022

Inhibition of translation termination by Drosocin, an antimicrobial peptide from fruit flies

A 19-amino acid long proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) is encoded in the fruit fly genome. Native Dro is glycosylated at a specific threonine residue, but the non-glycosylated peptide retains antibacterial activity. Dro shows sequence similarity to several other PrAMPs that bind in the ribosomal nascent peptide exit tunnel and inhibit protein synthesis by varying mechanisms. However, the target and mechanism of action of Dro remain unknown. Here we show that the primary mode of Dro action is inhibition of termination of protein synthesis. Our in vitro and in vivo experiments demonstrate that Dro stalls ribosomes at stop codons, likely sequestering class 1 release factors associated with the terminating ribosome. As the result, Dro strongly promotes readthrough of stop codons at subinhibitory concentrations. The elucidated mode of Dro action allows assigning it as the second member of the type II PrAMPs, of which only one representative, the antimicrobial peptide apidaecin (Api) produced by honeybees, was previously known. However, despite its functional similarity with Api, Dro interacts with the target in a markedly distinct way. The analysis of a comprehensive single-amino acid substitution library of endogenously expressed Dro variants shows that binding to the ribosome involves interactions of multiple amino acid residues distributed through the entire length of the PrAMP. Our data further show that the ribosome-targeting activity of non-glycosylated Dro can be significantly enhanced by single amino acid substitutions illuminating directions for improving its antibacterial properties.

molecular biology↗

bioRxiv2021

Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

Free L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

molecular biology↗

bioRxiv2020

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here, genome-wide analysis revealed that Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate development of new medicines and research tools for genome exploration.

biochemistry↗

bioRxiv2019

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the alternative bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

molecular biology↗