Biology subjects

Keiler, K. C.

Publications and source records attributed to Keiler, K. C..

11 recordsLinked to original sources

bioRxiv2026

Molecular sterics guide tmRNA translocation on the ribosome

Translation of defective mRNA transcripts can lead to the formation of stalled ``non-stop'' ribosome complexes. To rescue these ribosomes, bacteria have evolved a mechanism called trans-translation that is mediated by transfer-messenger RNA (tmRNA) and small protein B (SmpB). During the translocation step of trans-translation, the stalled mRNA is ejected and the reading frame found in the mRNA-like domain (MLD) of tmRNA is loaded into the mRNA channel. The ribosome then translates the new template, thereby tagging the nascent protein for degradation. While structures of tmRNA-SmpB on the ribosome have been available for years, little is known about the molecular factors that govern the dynamics of tmRNA translocation. Here, we use an energetically smooth model (i.e. SMOG) to simulate translocation of tmRNA-SmpB, which reveals that the structure of the ribosome gives rise to a preferred sequence of conformational substeps. Specifically, the tRNA-like domain (TLD) translocates, then the MLD loads, and finally the C-terminal tail of SmpB rearranges. These steps are interdependent and associated with a range of sterically-induced barriers that regulate the kinetics. This provides a structural and energetic framework for understanding, and ultimately controlling, the dynamics of trans-translation.

biophysics↗

bioRxiv2026

An RNase Toxin Hijacks Elongation Factor-Tu to Cleave the Ribosomal Sarcin-Ricin Loop

Ribosome-targeting toxins typically recognize and attack their substrates directly. Here, we provide the functional characterization of SRLase1, a widespread member of a recently identified RNase superfamily, and show that it cleaves the sarcin-ricin loop (SRL) of 23S rRNA. Unlike previously described ribosome-targeting toxins, SRLase1 requires the host elongation factor Tu (EF-Tu) for activity. EF-Tu normally engages the ribosome as part of the aminoacyl-tRNA delivery cycle, but SRLase1 instead exploits this factor through a mechanism incompatible with canonical ternary-complex formation. This host-factor-assisted mechanism reveals a distinct strategy for translation inhibition, in which a toxin repurposes a conserved component of the elongation machinery to access a vulnerable ribosomal target. Homologs of SRLase1 are widespread across bacterial phyla and are frequently encoded in conflict-associated genomic loci. Together, these findings define a widespread family of SRL-targeting toxins, expand the mechanistic repertoire of ribosomal antibacterial toxins, and demonstrate how conserved components of the host translation machinery can be co-opted to promote toxin activity.

microbiology↗

bioRxiv2026

Blocked transcription-translation complexes are rescued by transcript release followed by trans-translation

Bacterial ribosomes initiate translation while the nascent transcript is still engaged with RNA polymerase, creating a risk that translating ribosomes will become trapped if RNA polymerase is blocked before transcription of the stop codon. Here we show that DNA-binding proteins block RNA polymerase and translating ribosomes in vitro and in vivo. Translating ribosomes are rescued after they trigger release of the nascent transcript from RNA polymerase. Following release of the transcript, ribosomes translate to the 3 end of the mRNA and are rescued by trans-translation. This mechanism allows rescue of all components of blocked transcription-translation reactions.

molecular biology↗

bioRxiv2026

trans-Translation inhibitors that kill M. tuberculosis and pathogenic Non-tuberculous Mycobacteria have a dual mechanism of action

Mycobacterium tuberculosis and pathogenic nontuberculous mycobacteria pose a growing challenge to human health, and new antibiotics that target new pathways with novel mechanisms of action are urgently needed. Acylaminooxadiazole derivatives have previously been shown to inhibit the trans-translation ribosome rescue pathway and kill M. tuberculosis. Here, we show that modifications to the acylaminooxadiazole scaffold can improve potency and tune mycobacterial species specificity, resulting in molecules that kill M. avium, M. abscessus, and M. tuberculosis clinical isolates. Free iron was previously shown to antagonize antibacterial activity and decrease the inhibition of trans-translation by acylaminooxadiazoles, but we found that biologically relevant iron sources such as hemin and transferrin do not affect activity. Mutants depleted for tmRNA and mutants defective in siderophore-mediated iron utilization are both hypersusceptible to acylaminooxadiazole-based trans-translation inhibitors, indicating a dual mechanism of action involving both direct inhibition of trans-translation and metal starvation. These findings establish acylaminooxadiazoles as dual-mechanism antimycobacterial agents that couple inhibition of trans-translation with disruption of iron homeostasis.

microbiology↗

bioRxiv2025

A trans-translation inhibitor kills Mycobacterium tuberculosis by targeting ribosomal protein bL12

New antibiotics with novel mechanisms of action are needed to treat infections by multidrug-resistant strains of Mycobacterium tuberculosis. Here, we show that KKL-1005, an anti-tubercular triazole-based molecule, binds to ribosomal protein bL12 and specifically inhibits the trans-translation ribosome rescue pathway, a process essential for the survival of M. tuberculosis. Our data demonstrate that KKL-1005 binds to the N terminal domain of bL12, both in vitro and in bacterial cells, and specifically inhibits trans-translation and not normal translation. These results suggest that tmRNA-SmpB interacts with bL12 differently from tRNA, and raise the possibility of developing antibiotics targeting bL12.

microbiology↗

bioRxiv2024

Trans-Translation inhibitors and copper ions synergize for enhanced antibiotic activity

Trans-Translation is the most effective ribosome rescue mechanism and a compelling target for new antimicrobial agents. A recent proteomic study revealed similarities between the responses of Bacillus subtilis to the inhibitors small-molecule inhibitors oxadiazole KKL-40 and tetrazole KKL-55 and divalent cation ionophores, indicating the disturbance of metal homeostasis as potential secondary mechanism of action. Here, we report increased copper levels in KKL-40 and KKL-55-treated B. subtilis. Both inhibitors form copper complexes that enter large unilamellar vesicles. Copper supplementation enhanced the antibacterial activity against B. subtilis by simultaneously increasing inhibitor and copper uptake. The co-treatment of B. subtilis with trans-translation inhibitors and copper concentrations normally benign for trans-translation-competent cells, caused an immediate stalling of growth and translation, as observed at higher KKL-40 and KKL-55 concentrations without copper supplementation. Proteome analysis showed that during translation stalling cells were unable to mount an effective copper toxicity response. Taken together, the synergetic uptake of KKL-40 and KKL-55 with copper leads to a quick-onset translation stalling, preventing B. subtilis from counteracting the toxic effects of rapid copper influx. Significance statementThe challenge of antimicrobial resistance is growing, necessitating an exploration of novel antibiotic targets. Among these, trans-translation has attracted considerable attention due to its ubiquitous presence in bacteria as well as its role in virulence and pathogenesis. Several inhibitors of trans-translation have been identified in a target-based screening using a whole-cell assay. However, recent proteomic profiling studies suggested that the tested trans-translation inhibitors might have an additional mode of action. In this work, we shed light on their previously undiscovered copper ionophore activity and explore the consequences of co-treating B. subtilis with KKL-40 or KKL-55 and CuCl2. This co-treatment results in a rapid antibiotic influx, and, consequently to the stalling of ribosomes, translation, and bacterial growth. Simultaneously, massive amounts of copper accumulate in the cells, the toxic effects of which require a copper stress response to mitigate. However, such a response is averted by the stalled translation. Dual mechanism antibacterial agents are attractive because they are typically associated with slow emergence of resistance. A deep understanding of the complex interplay of KKL-40 and KKL-55 with metal ions will help to fully exploit trans-translation as an antibacterial target and to develop KKL-40 and KKL-55-based antibiotics into novel treatments for bacterial infections.

microbiology↗

bioRxiv2024

A trans-translation inhibitor is potentiated by zinc and kills Mycobacterium tuberculosis and non-tuberculous mycobacteria

Mycobacterium tuberculosis poses a serious challenge for human health, and new antibiotics with novel targets are needed. Here we demonstrate that an acylaminooxadiazole, MBX-4132, specifically inhibits the trans-translation ribosome rescue pathway to kill M. tuberculosis. Our data demonstrate that MBX-4132 is bactericidal against multiple pathogenic mycobacterial species and kills M. tuberculosis in macrophages. We also show that acylaminooxadiazole activity is antagonized by iron but is potentiated by zinc. Our transcriptomic data reveals dysregulation of multiple metal homeostasis pathways after exposure to MBX-4132. Furthermore, we see differential expression of genes related to zinc sensing and efflux when trans-translation is inhibited. Taken together, these data suggest that there is a link between disturbing intracellular metal levels and acylaminooxadiazole-mediated inhibition of trans-translation. These findings provide an important proof-of-concept that trans-translation is a promising antitubercular drug target.

microbiology↗

bioRxiv2023

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA

Trans-Translation is conserved throughout bacteria and is essential in many species. High-throughput screening identified a tetrazole-based trans-translation inhibitor, KKL-55, that has broad-spectrum antibiotic activity. A biotinylated version of KKL-55 pulled down Elongation Factor Thermo-unstable (EF-Tu) from bacterial lysates. Purified EF-Tu bound KKL-55 in vitro with a Kd = 2 {micro}M, confirming a high-affinity interaction. An X-ray crystal structure showed KKL-55 binds in domain 3 of EF-Tu, and mutation of residues in the binding pocket abolished KKL-55 binding. RNA binding assays in vitro showed that KKL-55 inhibits binding between EF-Tu and tmRNA, but not between EF-Tu and tRNA. These data demonstrate a new mechanism for inhibition of EF-Tu function and suggest that this specific inhibition of EF-Tu*tmRNA binding is a viable target for antibiotic development. IMPORTANCEEF-Tu is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers tmRNA-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

biochemistry↗

bioRxiv2020

Ribosome rescue inhibitors clear Neisseria gonorrhoeae in vivo using a new mechanism

The trans-translation pathway for rescuing stalled ribosomes is conserved and essential in bacterial pathogens but has no mammalian homolog, making it an ideal target for new antibiotics. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, resulting in broad-spectrum antibiotic activity. Optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles produced MBX-4132, which cleared multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Cryo-EM studies of non-stop ribosomes showed that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein L27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. One Sentence SummaryRibosome rescue inhibitors reveal a new conformation of the ribosome and kill drug-resistant Neisseria gonorrhoeae in vivo.

microbiology↗

bioRxiv2020

Reproducible and accessible analysis of transposon insertion data at scale

Significant progress has been made in advancing and standardizing tools for human genomic and biomedical research, yet the field of next generation sequencing (NGS) analysis for microorganisms (including multiple pathogens) remains fragmented, lacks accessible and reusable tools, is hindered by local computational resource limitations, and does not offer widely accepted standards. One of such "problem areas" is the analysis of Transposon Insertion Sequencing (TIS) data. TIS allows perturbing the entire genome of a microorganism by introducing random insertions of transposon-derived constructs. The impact of the insertions on the survival and growth provides precise information about genes affecting specific phenotypic characteristics. A wide array of tools has been developed to analyze TIS data and among the variety of options available, it is often difficult to identify which one can provide a reliable and reproducible analysis. Here we sought to understand the challenges and propose reliable practices for the analysis of TIS experiments. Using data from two recent TIS studies we have developed a series of workflows that include multiple tools for data de-multiplexing, promoter sequence identification, transposon flank alignment, and read count repartition across the genome. Particular attention was paid to quality control procedures such as the determination of the optimal tool parameters for the analysis and removal of contamination. Our work provides an assessment of the currently available tools for TIS data analysis and offers ready to use workflows that can be invoked by anyone in the world using our public Galaxy platform (https://usegalaxy.org). To lower the entry barriers we have also developed interactive tutorials explaining details of TIS data analysis procedures at https://bit.ly/gxy-tis. ImportanceA wide array of tools has been developed to analyze TIS data and among the variety of options available, it is often difficult to identify which one can provide a reliable and reproducible analysis. Here we sought to understand the challenges and propose reliable practices for the analysis of TIS experiments. Using data from two recent TIS studies we have developed a series of workflows that include multiple tools for data de-multiplexing, promoter sequence identification, transposon flank alignment, and read count repartition across the genome. Particular attention was paid to quality control procedures such as the determination of the optimal tool parameters for the analysis and removal of contamination. Our work democratizes the TIS data analysis by providing open workflows supported by public computational infrastructure.

microbiology↗

bioRxiv2018

A new mechanism for ribosome rescue can recruit RF1 or RF2 to non-stop ribosomes

Bacterial ribosomes frequently translate to the 3 end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these non-stop ribosomes are rescued using trans-translation. In some species, genes required for trans-translation are essential, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type cells but not in cells that lack trans-translation activity. Over-expression of ArfT suppresses the slow growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues non-stop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on non-stop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.\n\nImportanceFrancisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with the greatest risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identifies a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

microbiology↗