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Dual reporters encoding two distinct proteins within the same mRNA have had a crucial role in identifying and characterizing unconventional mechanisms of eukaryotic translation. These mechanisms include initiation via internal ribosomal entry sites (IRESs), ribosomal frameshifting, stop codon readthrough and reinitiation. This design enables the expression of one reporter to be influenced by the specific mechanism under investigation, while the other reporter serves as an internal control. However, challenges arise when intervening test sequences are placed between these two reporters. Such sequences can inadvertently impact the expression or function of either reporter, independent of translation-related changes, potentially biasing the results. These effects may occur due to cryptic regulatory elements inducing or affecting transcription initiation, splicing, polyadenylation and antisense transcription as well as unpredictable effects of the translated test sequences on the stability and activity of the reporters. Unfortunately, these unintended effects may lead to misinterpretation of data and the publication of incorrect conclusions in the scientific literature. To address this issue and to assist the scientific community in accurately interpreting dual-reporter experiments, we have developed comprehensive guidelines. These guidelines cover experimental design, interpretation and the minimal requirements for reporting results. They are designed to aid researchers conducting these experiments as well as reviewers, editors and other investigators who seek to evaluate published data., Competing Interests: Competing interests: G.L. and P.V.B. are cofounders and shareholders of EIRNA Bio Ltd. The remaining authors declare no competing interests., (© 2025. Springer Nature America, Inc.)

The paenilamicins are a group of hybrid nonribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here we determine structures of paenilamicin PamB2-stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site transfer RNAs (tRNAs). In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms used by P. larvae. We further demonstrate that PamB2 interferes with the translocation of messenger RNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a class of context-specific translocation inhibitors., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Apidaecin 1b (Api), the first characterized Type II Proline-rich antimicrobial peptide (PrAMP), is encoded in the honey bee genome. It inhibits bacterial growth by binding in the nascent peptide exit tunnel of the ribosome after the release of the completed protein and trapping the release factors. By genome mining, we have identified 71 PrAMPs encoded in insect genomes as pre-pro-polyproteins. Having chemically synthesized and tested the activity of 26 peptides, we demonstrate that despite significant sequence variation in the N-terminal sequence, the majority of the PrAMPs that retain the conserved C-terminal sequence of Api are able to trap the ribosome at the stop codons and induce stop codon readthrough-all hallmarks of Type II PrAMP mode of action. Some of the characterized PrAMPs exhibit superior antibacterial activity in comparison with Api. The newly solved crystallographic structures of the ribosome complexed with Api and with the more active peptide Fva1 from the stingless bee demonstrate the universal placement of the PrAMPs' C-terminal pharmacophore in the post-release ribosome despite variations in their N-terminal sequence., (© 2024. The Author(s).)

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., Competing Interests: COMPETING INTERESTS STATEMENT The authors declare no competing interests.

The Proline-rich Antimicrobial Peptide (PrAMP) apidaecin (Api) inhibits translation by binding in the ribosomal nascent peptide exit tunnel, trapping release factors RF1 or RF2, and arresting ribosomes at stop codons. To explore the extent of sequence variations of the native 18-amino acid Api that allows it to preserve its activity, we screened a library of synthetic mutant Api genes expressed in bacterial cells, resulting in nearly 350000 peptide variants with multiple substitutions. By applying orthogonal negative and positive selection strategies, we identified a number of multi-substituted Api variants capable of arresting ribosomes at stop codons. Our findings underscore the critical contribution of specific amino acid residues of the peptide for its on-target function while significantly expanding the variety of PrAMPs acting on the terminating ribosome. Additionally, some of the tested synthesized multi-substituted Api variants exhibit improved antibacterial activity compared to that of the wild type PrAMP and may constitute the starting point to develop clinically useful antimicrobials., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus . While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae . We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors., Competing Interests: Competing interests The authors declare no competing interests.

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 TnaC-mediated 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Streptococcus pneumoniae, an opportunistic human pathogen, is the leading cause of community-acquired pneumonia and an agent of otitis media, septicemia, and meningitis. Although genomic and transcriptomic studies of S. pneumoniae have provided detailed perspectives on gene content and expression programs, they have lacked information pertaining to the translational landscape, particularly at a resolution that identifies commonly overlooked small open reading frames (sORFs), whose importance is increasingly realized in metabolism, regulation, and virulence. To identify protein-coding sORFs in S. pneumoniae, antibiotic-enhanced ribosome profiling was conducted. Using translation inhibitors, 114 novel sORFs were detected, and the expression of a subset of them was experimentally validated. Two loci associated with virulence and quorum sensing were examined in deeper detail. One such sORF, rio3 , overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio3 from srf-02 revealed that rio3 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in the genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome. IMPORTANCE This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae. We identified 114 unannotated intergenic small open reading frames (sORFs). The described procedures and data sets provide a model for microbiologists seeking to explore the translational landscape of bacteria. The biological roles of four sORF examples are characterized: two control the regulation of a cell-cell communication (quorum sensing) system, one contributes to the ability of S. pneumoniae to colonize the upper respiratory tract of mice, and a fourth governs the translation of PrfB, a protein enabling ribosome release at stop codons. We propose that Ribo-RET is a valuable approach to identifying unstudied microproteins and difficult-to-find pheromone genes used by Gram-positive organisms, whose genomes are replete with pheromone receptors.

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, 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 TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo 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. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite., (© 2021. The Author(s).)

Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics., (© 2021. The Author(s).)

Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide., Competing Interests: The authors declare no competing interest.

The knowledge of translation start sites is crucial for annotation of genes in bacterial genomes. However, systematic mapping of start codons in bacterial genes has mainly relied on predictions based on protein conservation and mRNA sequence features which, although useful, are not always accurate. We recently found that the pleuromutilin antibiotic retapamulin (RET) is a specific inhibitor of translation initiation that traps ribosomes specifically at start codons, and we used it in combination with ribosome profiling to map start codons in the Escherichia coli genome. This genome-wide strategy, that was named Ribo-RET, not only verifies the position of start codons in already annotated genes but also enables identification of previously unannotated open reading frames and reveals the presence of internal start sites within genes. Here, we provide a detailed Ribo-RET protocol for E. coli. Ribo-RET can be adapted for mapping the start codons of the protein-coding sequences in a variety of bacterial species.

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 reveals that in bacteria, 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 the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration., Competing Interests: KM, TF, XS, DK, IC, ZI, YG, AM, NV No competing interests declared, (© 2020, Mangano et al.)

5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.

Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide-bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not substantially alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site transfer RNA to undergo repeated rounds of dissociation and nonproductive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.

While most of the ribosome-targeting antibiotics are bacteriostatic, some members of the macrolide class demonstrate considerable bactericidal activity. We previously showed that an extended alkyl-aryl side chain is the key structural element determining the macrolides' slow dissociation from the ribosome and likely accounts for the antibiotics' cidality. In the nontranslating Escherichia coli ribosome, the extended side chain of macrolides interacts with 23S ribosomal RNA (rRNA) nucleotides A752 and U2609, that were proposed to form a base pair. However, the existence of this base pair in the translating ribosome, its possible functional role, and its impact on the binding and cidality of the antibiotic remain unknown. By engineering E. coli cells carrying individual and compensatory mutations at the 752 and 2609 rRNA positions, we show that integrity of the base pair helps to modulate the ribosomal response to regulatory nascent peptides, determines the slow dissociation rate of the extended macrolides from the ribosome, and increases their bactericidal effect. Our findings demonstrate that the ability of antibiotics to kill bacterial cells relies not only on the chemical nature of the inhibitor, but also on structural features of the target., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the 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 internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.

The ribosome is a major antibiotic target. Many types of inhibitors can stop cells from growing by binding at functional centers of the ribosome and interfering with its ability to synthesize proteins. These antibiotics were usually viewed as general protein synthesis inhibitors, which indiscriminately stop translation at every codon of every mRNA, preventing the ribosome from making any protein. However, at each step of the translation cycle, the ribosome interacts with multiple ligands (mRNAs, tRNA substrates, translation factors, etc.), and as a result, the properties of the translation complex vary from codon to codon and from gene to gene. Therefore, rather than being indiscriminate inhibitors, many ribosomal antibiotics impact protein synthesis in a context-specific manner. This review presents a snapshot of the growing body of evidence that some, and possibly most, ribosome-targeting antibiotics manifest site specificity of action, which is modulated by the nature of the nascent protein, the mRNA, or the tRNAs.

Macrolide antibiotics inhibit protein synthesis by targeting the bacterial ribosome. They bind at the nascent peptide exit tunnel and partially occlude it. Thus, macrolides have been viewed as 'tunnel plugs' that stop the synthesis of every protein. More recent evidence, however, demonstrates that macrolides selectively inhibit the translation of a subset of cellular proteins, and that their action crucially depends on the nascent protein sequence and on the antibiotic structure. Therefore, macrolides emerge as modulators of translation rather than as global inhibitors of protein synthesis. The context-specific action of macrolides is the basis for regulating the expression of resistance genes. Understanding the details of the mechanism of macrolide action may inform rational design of new drugs and unveil important principles of translation regulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Genetic coding in bacteria largely operates via the "one gene-one protein" paradigm. However, the peculiarities of the mRNA structure, the versatility of the genetic code, and the dynamic nature of translation sometimes allow organisms to deviate from the standard rules of protein encoding. Bacteria can use several unorthodox modes of translation to express more than one protein from a single mRNA cistron. One such alternative path is the use of additional translation initiation sites within the gene. Proteins whose translation is initiated at different start sites within the same reading frame will differ in their N termini but will have identical C-terminal segments. On the other hand, alternative initiation of translation in a register different from the frame dictated by the primary start codon will yield a protein whose sequence is entirely different from the one encoded in the main frame. The use of internal mRNA codons as translation start sites is controlled by the nucleotide sequence and the mRNA folding. The proteins of the alternative proteome generated via the "genes-within-genes" strategy may carry important functions. In this review, we summarize the currently known examples of bacterial genes encoding more than one protein due to the utilization of additional translation start sites and discuss the known or proposed functions of the alternative polypeptides in relation to the main protein product of the gene. We also discuss recent proteome- and genome-wide approaches that will allow the discovery of novel translation initiation sites in a systematic fashion.

Antibiotics can cause dormancy (bacteriostasis) or induce death (cidality) of the targeted bacteria. The bactericidal capacity is one of the most important properties of antibacterial agents. However, the understanding of the fundamental differences in the mode of action of bacteriostatic or bactericidal antibiotics, especially those belonging to the same chemical class, is very rudimentary. Here, by examining the activity and binding properties of chemically distinct macrolide inhibitors of translation, we have identified a key difference in their interaction with the ribosome, which correlates with their ability to cause cell death. While bacteriostatic and bactericidal macrolides bind in the nascent peptide exit tunnel of the large ribosomal subunit with comparable affinities, the bactericidal antibiotics dissociate from the ribosome with significantly slower rates. The sluggish dissociation of bactericidal macrolides correlates with the presence in their structure of an extended alkyl-aryl side chain, which establishes idiosyncratic interactions with the ribosomal RNA. Mutations or chemical alterations of the rRNA nucleotides in the drug binding site can protect cells from macrolide-induced killing, even with inhibitor concentrations that significantly exceed those required for cell growth arrest. We propose that the increased translation downtime due to slow dissociation of the antibiotic may damage cells beyond the point where growth can be reinitiated upon the removal of the drug due to depletion of critical components of the gene-expression pathway., Competing Interests: Conflict of interest statement: In the previous years, A.S.M. had grants from Cempra, Inc. and Melinta Therapeutics, which were involved in the development of macrolide antibiotics.

Antibiotics methymycin (MTM) and pikromycin (PKM), co-produced by Streptomyces venezuelae, represent minimalist macrolide protein synthesis inhibitors. Unlike other macrolides, which carry several side chains, a single desosamine sugar is attached to the macrolactone ring of MTM and PKM. In addition, the macrolactone scaffold of MTM is smaller than in other macrolides. The unusual structure of MTM and PKM and their simultaneous secretion by S. venezuelae bring about the possibility that two compounds would bind to distinct ribosomal sites. However, by combining genetic, biochemical and crystallographic studies, we demonstrate that MTM and PKM inhibit translation by binding to overlapping sites in the ribosomal exit tunnel. Strikingly, while MTM and PKM readily arrest the growth of bacteria, ∼40% of cellular proteins continue to be synthesized even at saturating concentrations of the drugs. Gel electrophoretic analysis shows that compared to other ribosomal antibiotics, MTM and PKM prevent synthesis of a smaller number of cellular polypeptides illustrating a unique mode of action of these antibiotics., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many antibiotics stop bacterial growth by inhibiting different steps of protein synthesis. However, no specific inhibitors of translation termination are known. Proline-rich antimicrobial peptides, a component of the antibacterial defense system of multicellular organisms, interfere with bacterial growth by inhibiting translation. Here we show that Api137, a derivative of the insect-produced antimicrobial peptide apidaecin, arrests terminating ribosomes using a unique mechanism of action. Api137 binds to the Escherichia coli ribosome and traps release factor (RF) RF1 or RF2 subsequent to the release of the nascent polypeptide chain. A high-resolution cryo-EM structure of the ribosome complexed with RF1 and Api137 reveals the molecular interactions that lead to RF trapping. Api137-mediated depletion of the cellular pool of free release factors causes the majority of ribosomes to stall at stop codons before polypeptide release, thereby resulting in a global shutdown of translation termination.

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center., Competing Interests: The research in the laboratory was supported by, among other sources, grants from the pharmaceutical companies Melinta Therapeutics and Cempra Pharmaceuticals.

Macrolide antibiotic binding to the ribosome inhibits catalysis of peptide bond formation between specific donor and acceptor substrates. Why particular reactions are problematic for the macrolide-bound ribosome remains unclear. Using comprehensive mutational analysis and biochemical experiments with synthetic substrate analogs, we find that the positive charge of these specific residues and the length of their side chains underlie inefficient peptide bond formation in the macrolide-bound ribosome. Even in the absence of antibiotic, peptide bond formation between these particular donors and acceptors is rather inefficient, suggesting that macrolides magnify a problem present for intrinsically difficult substrates. Our findings emphasize the existence of functional interactions between the nascent protein and the catalytic site of the ribosomal peptidyl transferase center., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulation of gene expression in response to the changing environment is critical for cell survival. For instance, binding of macrolide antibiotics to the ribosome promotes translation arrest at the leader open reading frames ermCL and ermBL, which is necessary for inducing the antibiotic resistance genes ermC and ermB. Cladinose-containing macrolides such as erythromycin (ERY), but not ketolides such as telithromycin (TEL), arrest translation of ermCL, whereas either ERY or TEL stall ermBL translation. How the ribosome distinguishes between chemically similar small molecules is unknown. We show that single amino acid changes in the leader peptide switch the specificity of recognition of distinct molecules, triggering gene activation in response to ERY alone, to TEL alone or to both antibiotics or preventing stalling altogether. Thus, the ribosomal response to chemical signals can be modulated by minute changes in the nascent peptide, suggesting that protein sequences could have been optimized for rendering translation sensitive to environmental cues.

Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism.

Negamycin (NEG) is a ribosome-targeting antibiotic that exhibits clinically promising activity. Its binding site and mode of action have remained unknown. We solved the structure of the Thermus thermophilus ribosome bound to mRNA and three tRNAs, in complex with NEG. The drug binds to both small and large ribosomal subunits at nine independent sites. Resistance mutations in the 16S rRNA unequivocally identified the binding site in the vicinity of the conserved helix 34 (h34) in the small subunit as the primary site of antibiotic action in the bacterial and, possibly, eukaryotic ribosome. At this site, NEG contacts 16S rRNA as well as the anticodon loop of the A-site tRNA. Although the NEG site of action overlaps with that of tetracycline (TET), the two antibiotics exhibit different activities: while TET sterically hinders binding of aminoacyl-tRNA to the ribosome, NEG stabilizes its binding, thereby inhibiting translocation and stimulating miscoding., (Copyright © 2014 Elsevier Inc. All rights reserved.)

During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9 Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site, leading to inhibition of peptide bond formation and translation arrest., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Translation arrest directed by nascent peptides and small cofactors controls expression of important bacterial and eukaryotic genes, including antibiotic resistance genes, activated by binding of macrolide drugs to the ribosome. Previous studies suggested that specific interactions between the nascent peptide and the antibiotic in the ribosomal exit tunnel play a central role in triggering ribosome stalling. However, here we show that macrolides arrest translation of the truncated ErmDL regulatory peptide when the nascent chain is only three amino acids and therefore is too short to be juxtaposed with the antibiotic. Biochemical probing and molecular dynamics simulations of erythromycin-bound ribosomes showed that the antibiotic in the tunnel allosterically alters the properties of the catalytic center, thereby predisposing the ribosome for halting translation of specific sequences. Our findings offer a new view on the role of small cofactors in the mechanism of translation arrest and reveal an allosteric link between the tunnel and the catalytic center of the ribosome.

Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li et al. gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the functional needs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In bacteria, ribosome stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here we present a cryo-electron microscopy structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase centre (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural basis for understanding how the drug and the nascent peptide cooperate to inhibit peptide bond formation and induce translation arrest.