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.

Proline-rich antimicrobial peptides (PrAMPs) kill bacteria by binding in the ribosomal nascent peptide exit tunnel. Type II PrAMPs bind in an orientation matching that of the nascent protein, trap the release factors and arrest ribosomes at stop codons. Conversely, Type I PrAMPs bind in an opposite orientation: their N-terminus invades the peptidyl transferase center arresting translation at start codons. Here, by mining the genome databases, we identify a number of PrAMPs with high sequence similarity to the Type II PrAMP Drosocin. Notably, many of the new PrAMPs do not stall ribosomes at stop codons, but act as Type I PrAMPs arresting translation at start codons. Structural analysis shows that such peptides bind with a Type I orientation. Minimal alterations in the peptide structure can flip the orientation of the PrAMP in the exit tunnel, switching the mechanism of translation inhibition. Altering the mode of binding and action of a PrAMP by only few mutations could be exploited by the host to combat newly emerging bacterial pathogens.

Type II proline-rich antimicrobial peptides have emerged as promising therapeutic candidates due to their ability to inhibit a previously undrugged stage of bacterial protein synthesis: translation termination. The recent characterization of the potent Type II proline-rich antimicrobial peptide, Pdi1, suggests that it may represent a promising therapeutic candidate; however, no information is available concerning its ribosome-binding activity. The lack of biochemical and biophysical assays to interrogate the binding affinities and mechanism of action of this class of peptides presents a bottleneck in the drug-discovery pipeline. In this study, we report the first characterization of the ribosome-binding kinetics of a Type II proline-rich antimicrobial peptide, for which we applied a biolayer interferometry assay to study the Pdi1-ribosome interaction. Our results reveal slow dissociation kinetics, which indicates prolonged residence time in the ribosomal nascent peptide exit tunnel. Additionally, alanine scanning of the Pdi1 C-terminus revealed that specific residues are critical for ribosome binding and contribute to the rate of Pdi1 dissociation. These findings were validated in cell-based assays where alanine substitutions at these positions reduced antibiotic activity. We also demonstrated that a Pdi1 derivative, which lacks the third-to-last pharmacophoric residue, fails to inhibit translation termination, despite retaining a submicromolar minimum inhibitory concentration. These results demonstrate that select residues in the Pdi1 pharmacophore play a critical role in ribosome binding and dissociation, in addition to inhibiting translation termination.

Cellular protein synthesis relies on random encounters between ribosomes and mRNAs, limiting optimization of the translation machinery for production of a single protein-a key need in biotechnology. One potential solution is integrating the protein-coding sequence into the ribosome itself, thereby committing the ribosome to synthesis of a single polypeptide. The feasibility of such integration could also address a long-standing challenge in RNA world models: explaining how early protein synthesis could function reliably despite the scarcity and poor organization of its components1. Whether a ribosome can translate its own ribosomal RNA (rRNA) has remained unclear. Here we show that bacterial ribosomes can synthesize proteins encoded within their own RNA. We engineered a chimeric messenger-ribosomal RNA (mrRNA) by appending a protein-coding sequence to 16S rRNA. The hybrid mrRNA assembles into a small ribosomal subunit that binds to the large subunit to form Ribo-M, a ribosome capable of translating mrRNA-encoded proteins. Translation is abolished by mutations or antibiotics that impair the function of the small subunit, demonstrating that mrRNA translation is carried out in cis by ribosomes assembled on the chimeric mrRNA. Incorporating mrRNA into a ribosome with tethered subunits yielded Ribo-TM, in which encoding, decoding and peptide synthesis are united within a single RNA scaffold. These findings establish the mechanistic feasibility of a ribosome translating its own rRNA in vivo and in vitro, offering a versatile platform for orthogonal protein production and insights into the origin of translation.

A key challenge in addressing the antibiotic resistance crisis is identifying new antimicrobial compounds1. Although natural products produced by fungi and bacteria, particularly actinomycetes, have been the source of most antibiotics discovered over the past 80 years, they have fallen out of favour owing to the frequent rediscovery of known drug scaffolds2. The current perception is that antibiotic-producing actinomycetes have been over-mined and possess little novelty left to yield. Here we demonstrate that by using improved fractionation approaches that enrich previously overlooked minor products, even well-studied strains of antibiotic-producing actinomycetes can provide new chemical scaffolds with unique modes of action. By fractionating a library of natural product extracts from soil bacteria, we show that Streptomyces rimosus, the source of the well-known antibiotic oxytetracycline, produces a cyclic depsipeptide antibiotic that we call manikomycin. Manikomycin can kill multidrug-resistant Enterobacteriaceae and is not susceptible to resistance associated with clinically used antibiotics. Biochemical, genetic and structural analyses reveal that manikomycin binds in the E-site of the large subunit of the bacterial ribosome, preventing entry of the 3 ' end of the tRNA into the E-site and effectively hindering the translocation step of protein synthesis in a sequence-context-specific manner. Manikomycin is the first antibacterial agent, to our knowledge, to target the critical but underexplored E-site in the large ribosomal subunit, highlighting its value as a lead for developing new antibiotics.

Tuberculosis is the deadliest bacterial disease on the planet. The months-long regimen of multiple antibiotics required to treat tuberculosis profoundly affects the microbiome and leads to the development of antimicrobial resistance. Furthermore, non-tuberculous mycobacterial infections pose an increasing clinical challenge. Consequently, there is a growing need for new narrow-spectrum mycobacteria-targeting antibiotics with different mechanisms of action. Here, we report the discovery and characterization of a natural glycolipid antibiotic, saskemycin (SKM), which demonstrates potent and highly selective activity against mycobacteria. Genome sequencing, chemical analysis, and isotope feeding strategies reveal the unique structure and biosynthetic origin of SKM. SKM binds to the small ribosomal subunit at a site not targeted by any of the clinically relevant antibiotics acting on the ribosome. Bound to the ribosome, SKM corrupts the decoding center in a unique way, preventing stable binding of aminoacyl-tRNA in the A site and inhibiting translation in a sequence context-specific manner. Self-resistance in the producing organism is conferred by methylation of a single 16S rRNA nucleotide by SasO and SasN rRNA methyltransferases. These enzymes are orthologs of the ubiquitous RsmC and SpoU methyltransferases found in most bacterial genera but absent in mycobacteria, rationalizing SKM's exquisite selectivity. The discovery of SKM provides an entry point for the development of selective, microbiome-sparing antimycobacterial antibiotics with a unique structure, binding site, and mechanism of action.

Lasso peptides, biologically active molecules with a distinct structurally constrained knotted fold, are natural products belonging to the class of ribosomally-synthesized and posttranslationally modified peptides (RiPPs). Lasso peptides act upon several bacterial targets, but none have been reported to inhibit the ribosome, one of the main antibiotic targets in the bacterial cell. Here, we report the identification and characterization of the lasso peptide antibiotic, lariocidin (LAR), and its internally cyclized derivative, lariocidin B (LAR-B), produced by Paenabacillussp. M2, with broad-spectrum activity against many bacterial pathogens. We show that lariocidins inhibit bacterial growth by binding to the ribosome and interfering with protein synthesis. Structural, genetic, and biochemical data show that lariocidins bind at a unique site in the small ribosomal subunit, where they interact with the 16S rRNA and aminoacyl-tRNA, inhibiting translocation and inducing miscoding. LAR is unaffected by common resistance mechanisms, has a low propensity for generating spontaneous resistance, shows no human cell toxicity, and has potent in vivo activity in a mouse model of Acinetobacter baumannii infection. Our finding of the first ribosome-targeting lasso peptides uncovers new routes toward discovering alternative protein synthesis inhibitors and offers a new chemical scaffold for developing much-needed antibacterial drugs.

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.

A significant challenge in addressing the antibiotic resistance crisis is identifying new antimicrobial compounds. Although natural products produced by fungi and bacteria, particularly actinomycetes, have been the source of most antibiotics discovered over the past 80 years, they have fallen out of favor due to the frequent rediscovery of known drug scaffolds. The current perception is that antibiotic-producing actinomycetes have been over-mined and possess little novelty left to yield. Here, we demonstrate that by using improved fractionation approaches that enrich previously overlooked minor products, even well-studied strains of antibiotic-producing actinomycetes can provide new chemical scaffolds with unique modes of action. By fractionating a library of natural product extracts from soil bacteria, we show that Streptomyces rimosus, the source of the well-known antibiotic oxytetracycline, produces a previously overlooked cyclic depsipeptide antibiotic that we called manikomycin. Manikomycin can kill multi-drug-resistant Enterobacteriaceae and is not susceptible to resistance associated with clinically used antibiotics. Biochemical, genetic, and structural analyses reveal that manikomycin binds in the 'exit' or E site of the large subunit of the bacterial ribosome preventing the entry of the 3' end of the tRNA into the E site and effectively hindering the translocation step of protein synthesis in a sequence context-specific manner. Manikomycin is the first antibacterial agent to target the critical but underexplored E site in the large ribosomal subunit, highlighting its value as a lead for developing new antibiotics.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts. Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis. Here, the authors show that the honey-bee derived PrAMPs Api137 and Api88 inhibit bacterial ribosomes through multiple mechanisms, promising for drug development.

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaCmediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

Growing resistance toward ribosome-targeting macrolide antibiotics has limited their clinical utility and urged the search for superior compounds. Macrolones are synthetic macrolide derivatives with a quinolone side chain, structurally similar to DNA topoisomerase-targeting fluoroquinolones. While macrolones show enhanced activity, their modes of action have remained unknown. Here, we present the first structures of ribosome-bound macrolones, showing that the macrolide part occupies the macrolide-binding site in the ribosomal exit tunnel, whereas the quinolone moiety establishes new interactions with the tunnel. Macrolones efficiently inhibit both the ribosome and DNA topoisomerase in vitro. However, in the cell, they target either the ribosome or DNA gyrase or concurrently both of them. In contrast to macrolide or fluoroquinolone antibiotics alone, dual-targeting macrolones are less prone to select resistant bacteria carrying target-site mutations or to activate inducible macrolide resistance genes. Furthermore, because some macrolones engage Erm-modified ribosomes, they retain activity even against strains with constitutive erm resistance genes. Hybrids of macrolides and quinolones, called macrolones, can overcome macrolide-induced resistance through new interactions between the quinolone moiety and the ribosome and can concurrently inhibit both ribosome and DNA gyrase targets.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).

With the growing crisis of antimicrobial resistance, it is critical to continue to seek out new sources of novel antibiotics. This need has led to renewed interest in natural product antimicrobials, specifically antimicrobial peptides. Nonlytic antimicrobial peptides are highly promising due to their unique mechanisms of action. One such peptide is apidaecin (Api), which inhibits translation termination through stabilization of the quaternary complex of the ribosome-apidaecin-tRNA-release factor. Synthetic derivatives of apidaecin have been developed, but structure-guided modifications have yet to be considered. In this work, we have focused on modifying key residues in the Api sequence that are responsible for the interactions that stabilize the quaternary complex. We present one of the first examples of a highly modified Api peptide that maintains its antimicrobial activity and interaction with the translation complex. These findings establish a starting point for further structure-guided optimization of Api peptides.

The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.

The research article describing the discovery of ribosomal frameshifting in the bacterial CopA gene also reported the occurrence of frameshifting in the expression of the human ortholog ATP7B based on assays using dual luciferase reporters. An examination of the publicly available ribosome profiling data and the phylogenetic analysis of the proposed frameshifting site cast doubt on the validity of this claim and prompted us to reexamine the evidence. We observed similar apparent frameshifting efficiencies as the original authors using the same type of vector that synthesizes both luciferases as a single polyprotein. However, we noticed anomalously low absolute luciferase activities from the N-terminal reporter that suggests interference of reporter activity or levels by the ATP7B test cassette. When we tested the same proposed ATP7B frameshifting cassette in a more recently developed reporter system in which the reporters are released without being included in a polyprotein, no frameshifting was detected above background levels.

Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.