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During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform β-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

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

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.HIGHLIGHTSThermorubin is a potent inhibitor of protein synthesis both in vivo and in vitro;Thermorubin does not prevent the binding of P- and A-site tRNAs;Thermorubin affects multiple steps of translation elongation with a major impact on binding stability of the A-site tRNA;Thermorubin can act as an inhibitor of translation termination on some ORFs.

Many antibiotics inhibit bacterial growth by binding to the ribosome and interfering with protein biosynthesis. Macrolides represent one of the most successful classes of ribosome-targeting antibiotics. The main clinically relevant mechanism of resistance to macrolides is dimethylation of the 23S rRNA nucleotide A2058, located in the drug-binding site, a reaction catalyzed by Erm-type rRNA methyltransferases. Here, we present the crystal structure of the Erm-dimethylated 70S ribosome at 2.4 A resolution, together with the structures of unmethylated 70S ribosome functional complexes alone or in combination with macrolides. Altogether, our structural data do not support previous models and, instead, suggest a principally new explanation of how A2058 dimethylation confers resistance to macrolides. Moreover, high-resolution structures of two macrolide antibiotics bound to the unmodified ribosome reveal a previously unknown role of the desosamine moiety in drug binding, laying a foundation for the rational knowledge-based design of macrolides that can overcome Erm-mediated resistance. Structural analysis of the A2058-dimethylated and unmethylated 70S ribosome complex alone and in combination with macrolides reveals the role of the desosamine moiety of macrolides in drug binding and resistance.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials, however their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, by X-ray crystallography and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Staphylococcus aureus. Two peptides out of five, acquired a weak membrane-perturbing activity, while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat the antibiotic-resistant pathogens.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site.

Summary Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.

Class I release factors (RFs) recognize stop codons in the sequences of mRNAs and are required for the hydrolysis of peptidyl-tRNA in the ribosomal P site during the final step of protein synthesis in bacteria, resulting in the release of a complete polypeptide chain from the ribosome. A key role in this process belongs to the highly conserved GGQ motif in RFs. Mutations in this motif can reduce the hydrolysis rate or even completely inhibit the reaction. Previously, it was hypothesized that the amino acid residues of GGQ (especially glutamine) are essential for the proper coordination of the water molecule for subsequent hydrolysis of the ester bond. However, available structures of the 70S ribosome termination complex do not allow unambiguous identification of the exact orientation of the carbonyl group in peptidyl-tRNA relative to the GGQ, as well as of the position of the catalytic water molecule in the peptidyl transferase center (PTC). This mini-review summarizes key facts and hypotheses on the role of GGQ in the catalysis of peptide release, as well as suggests and discusses future experiments aimed to produce high-quality structural data for deciphering the precise mechanism of RF-mediated catalysis.

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF) that confer resistance to oxazolidinone, such as linezolid, and phenicol antibiotics, such as chloramphenicol. PoxtA/OptrA are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria. These target protection proteins are thought to confer resistance by binding to the ribosome and dislodging the antibiotics from their binding sites. However, a structural basis for their mechanism of action has been lacking. Here we present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome at 2.9–3.1 Å, as well as the complete E. faecalis 70S ribosome at 2.2–2.5 Å. The structures reveal that PoxtA binds within the ribosomal E-site with its antibiotic resistance domain (ARD) extending towards the peptidyltransferase center (PTC) on the large ribosomal subunit. At its closest point, the ARD of PoxtA is still located >15 Å from the linezolid and chloramphenicol binding sites, suggesting that drug release is elicited indirectly. Instead, we observe that the ARD of PoxtA perturbs the CCA-end of the P-site tRNA causing it to shift by ∼4 Å out of the PTC, which correlates with a register shift of one amino acid for the attached nascent polypeptide chain. Given that linezolid and chloramphenicol are context-specific translation elongation inhibitors, we postulate that PoxtA/OptrA confer resistance to oxazolidinones and phenicols indirectly by perturbing the P-site tRNA and thereby altering the conformation of the attached nascent chain to disrupt the drug binding site.

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNA Phe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNA Val reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4’-deoxy-4’-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or ribosomal RNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

The 70S ribosome is a major target for antibacterial drugs. Two of the classical antibiotics, chloramphenicol (CHL) and erythromycin (ERY), competitively bind to adjacent but separate sites on the bacterial ribosome: the catalytic peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), respectively. The previously reported competitive binding of CHL and ERY might be due either to a direct collision of the two drugs on the ribosome or due to a drug-induced allosteric effect. Because of the resolution limitations, the available structures of these antibiotics in complex with bacterial ribosomes do not allow us to discriminate between these two possible mechanisms. In this work, we have obtained two crystal structures of CHL and ERY in complex with the Thermus thermophilus 70S ribosome at a higher resolution (2.65 and 2.89 Å, respectively) allowing unambiguous placement of the drugs in the electron density maps. Our structures provide evidence of the direct collision of CHL and ERY on the ribosome, which rationalizes the observed competition between the two drugs.

Transcriptional enhancers in the cell nuclei typically interact with the target promoters in cis over long stretches of chromatin, but the mechanism of this communication remains unknown. Previously we have developed a defined in vitro system for quantitative analysis of the rate of distant enhancer-promoter communication (EPC) and have shown that the chromatin fibers maintain efficient distant EPC in cis. Here we investigate the roles of linker histone H1 and HMGN5 protein in EPC. A considerable negative effect of histone H1 on EPC depending on its C- and N-tails was shown. Protein HMGN5 that affects chromatin compaction and is associated with active chromatin counteracts EPC inhibition by H1. The data suggest that the efficiency of the interaction between the enhancer and the promoter depends on the structure and dynamics of the chromatin fiber localized between them and can be regulated by proteins associated with chromatin.

During protein synthesis, ribosomes discriminate chirality of amino acids and prevent incorporation of D-amino acids into nascent proteins by slowing down the rate of peptide bond formation. Despite this phenomenon being known for nearly forty years, no structures have ever been reported that would explain the poor reactivity of D-amino acids. Here we report a 3.7Å-resolution crystal structure of a bacterial ribosome in complex with a D-aminoacyl-tRNA analog bound to the A site. Although at this resolution we could not observe individual chemical groups, we could unambiguously define the positions of the D-amino acid side chain and the amino group based on chemical restraints. The structure reveals that similarly to L-amino acids, the D-amino acid binds the ribosome by inserting its side chain into the ribosomal A-site cleft. This binding mode does not allow optimal nucleophilic attack of the peptidyl-tRNA by the reactive α-amino group of a D-amino acid. Also, our structure suggests that the D-amino acid cannot participate in hydrogen-bonding with the P-site tRNA that is required for the efficient proton transfer during peptide bond formation. Overall, our work provides the first mechanistic insight into the ancient mechanism that helps living cells ensure the stereochemistry of protein synthesis.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern1. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings2. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the $${\text{m}}_{2}^{6}\text{A}2058$$ nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance. Structure-guided design and component-based synthesis are used to produce iboxamycin, a novel ribosome-binding antibiotic with potent activity against Gram-positive and Gram-negative bacteria.

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern. For more than five decades, the search for new antibiotics has relied heavily upon the chemical modification of natural products (semi-synthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semi-synthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings. Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, here named iboxamycin. Iboxamycin is effective in strains expressing Erm and Cfr rRNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins, and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native 70S bacterial ribosome, as well as the Erm-methylated 70S ribosome, uncover the structural basis for this enhanced activity, including an unforeseen and unprecedented displacement of upon antibiotic binding. In mice, iboxamycin is orally bioavailable, safe, and effective in treating bacterial infections, testifying to the capacity for chemical synthesis to provide new antibiotics in an era of rising resistance.

Significance Sarecycline is the first narrow-spectrum tetracycline-class antibiotic that was recently approved by the FDA for the clinical treatment of acne vulgaris. In this work, we determined two (2.8-Å and 3.0-Å) X-ray crystal structures of sarecycline bound to the initiation complex of the bacterial 70S ribosome and found that this antibiotic inhibits bacterial ribosome in part using a mechanism of direct mRNA contact, which has not been reported for any other tetracyclines so far. Moreover, our structural analysis rationalizes why sarecycline is able to overcome one of the most common mechanisms of resistance to tetracyclines among pathogenic bacteria. Thus, this work provides mechanistic insights into the function of the tetracycline class of antibiotics on the ribosome with direct clinical relevance., Sarecycline is a new narrow-spectrum tetracycline-class antibiotic approved for the treatment of acne vulgaris. Tetracyclines share a common four-ring naphthacene core and inhibit protein synthesis by interacting with the 70S bacterial ribosome. Sarecycline is distinguished chemically from other tetracyclines because it has a 7-[[methoxy(methyl)amino]methyl] group attached at the C7 position of ring D. To investigate the functional role of this C7 moiety, we determined the X-ray crystal structure of sarecycline bound to the Thermus thermophilus 70S ribosome. Our 2.8-Å resolution structure revealed that sarecycline binds at the canonical tetracycline binding site located in the decoding center of the small ribosomal subunit. Importantly, unlike other tetracyclines, the unique C7 extension of sarecycline extends into the messenger RNA (mRNA) channel to form a direct interaction with the A-site codon to possibly interfere with mRNA movement through the channel and/or disrupt A-site codon–anticodon interaction. Based on our biochemical studies, sarecycline appears to be a more potent initiation inhibitor compared to other tetracyclines, possibly due to drug interactions with the mRNA, thereby blocking accommodation of the first aminoacyl transfer RNA (tRNA) into the A site. Overall, our structural and biochemical findings rationalize the role of the unique C7 moiety of sarecycline in antibiotic action.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3′ of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3′ pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTt NTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation. [Figure not available: see fulltext.], SCOPUS: ar.j, info:eu-repo/semantics/published

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. Identification of all enzymes responsible for rRNA methylation, as well as mapping of all modified rRNA residues, is now complete for a number of model species, such as Escherichia coli and Saccharomyces cerevisiae. Recent high-resolution structures of bacterial ribosomes provided the first direct visualization of methylated nucleotides. The structures of ribosomes from various organisms and organelles have also lately become available, enabling comparative structure-based analysis of rRNA methylation sites in various taxonomic groups. In addition to the conserved core of modified residues in ribosomes from the majority of studied organisms, structural analysis points to the functional roles of some of the rRNA methylations, which are discussed in this Review in an evolutionary context.

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 emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of the tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.

Communication between distantly spaced genomic regions is one of the key features of gene regulation in eukaryotes. Chromatin per se can stimulate efficient enhancer-promoter communication (EPC); however, the role of chromatin structure and dynamics in this process remains poorly understood. Here we show that nucleosome spacing and the presence of nucleosome-free DNA regions can modulate chromatin structure/dynamics and, in turn, affect the rate of EPC in vitro and in silico. Increasing the length of internucleosomal linker DNA from 25 to 60 bp results in more efficient EPC. The presence of longer nucleosome-free DNA regions can positively or negatively affect the rate of EPC, depending upon the length and location of the DNA region within the chromatin fiber. Thus the presence of histone-free DNA regions can differentially affect the efficiency of EPC, suggesting that gene regulation over a distance could be modulated by changes in the length of internucleosomal DNA spacers.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly expanding and largely untapped class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display an outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium sp. Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) – an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants belonging to the Rhizobiales. PHZ inhibits prokaryotic translation through the obstruction of the passage of the nascent peptide through the ribosome exit channel. The cryo-EM structure of the Escherichia coli ribosome with bound PHZ revealed that the drug interacts with the 23S rRNA and ribosomal proteins uL4 and uL22 and obstructs the exit tunnel in a way that is distinct from other compounds blocking the exit channel. We show that the sequence of uL4 ribosomal protein loop involved in PHZ binding determines the species-specificity of antibiotic interaction with its target. PHZ and its predicted homologs from other bacterial species expand the known diversity of LAPs and may be used in the future as biocontrol agents for the needs of agriculture.

Bifunctional Rel stringent factors, the most broadly distributed class of RSHs, are ribosome-associated enzymes that transfer a pyrophosphate group from ATP onto the 3′ of GTP or GDP to synthesize (p)ppGpp and also catalyse the 3′ pyrophosphate hydrolysis of the alarmone to degrade it. The precise regulation of these enzymes seems to be a complex allosteric mechanism, and despite decades of research, it is unclear how the two opposing activities of Rel are controlled at the molecular level. Here we show that a stretch/recoil guanosine-switch mechanism controls the catalytic cycle of T. thermophilus Rel (RelTf). The binding of GDP/ATP stretches apart the NTD catalytic domains of RelTf (RelTtNTD) activating the synthetase domain and allosterically blocking the hydrolase active site. Conversely, binding of ppGpp unlocks the hydrolase domain and triggers recoil of both NTDs, which partially buries the synthetase active site and precludes the binding of synthesis precursors. This allosteric mechanism acts as an activity switch preventing futile cycles of alarmone synthesis and degradation.

Background: About 50% of antibiotics used in the therapy of infectious diseases target bacterial 70S ribosomes. High resolution X-ray crystallographic studies allow us for determination of position of drug on the ribosome, but to elucidate the detailed molecular mechanism of inhibition it is necessary to study the dynamics of partial reactions of protein biosynthesis. Majority of high resolution structures were obtained on ribosomes from thermophilic bacteria T. thermophilus, whereas most of the functional studies were performed on reconstituted in vitro translation system from mesophilic organism E. coli. Despite of high homology among bacterial ribosomes, in many cases particular contacts observed are specific to thermophilic organism and is not allowing us to generalize the molecular mechanism of inhibition. Methods: Functional ribosomal pre-translocation complexes containing deacylated tRNAfMet in the P site and fMetPhe-tRNAPhe or fMetVal-tRNAVal in the A site were incubated with specific inhibitors: spectinomycin, amicoumacin, dirithromycin. Functional and structural studies were performed by using pre-steady state stopped-flow fluorescent spectroscopy and time-resolved cryo-electron microscopy. Kinetic studies of partial reactions of elongation were performed with thermophilic elongation factors and mesophilic reconstituted translation system. Results: Combination of structural data and pre-steady state kinetics reveals the details of molecular mechanism of inhibition of EF-G catalysed translocation and shows the intermediate conformations of ribosome-tRNA complexes during forward translocation. Heterologous translation system with substituted thermophilic elongation factors allows for differential studies of elongation cycle. Conclusion: Time-resolved high resolution cryo-electron microscopy is a method of choice for structural characterization of active complexes in physiological conditions in the process of functioning. Reconstituted in vitro translation system from E. coli can be used both for structural and functional studies, allowing merging of two types of data for extensive characterization of bacterial protein synthesis apparatus.

Objectives\ud \ud Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.\ud \ud \ud Methods\ud \ud A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.\ud \ud \ud Results\ud \ud MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.\ud \ud \ud Conclusions\ud \ud Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) – an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs., The authors report the identification of phazolicin (PHZ) - a prokaryotic translation inhibitory peptide - and its structure in complex with the E. coli ribosome, delineating PHZ’s mode of action and suggesting a basis for its bacterial species-specific activity.

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin–RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome—the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity.

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. Therefore, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.

International audience; Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

Antibiotic chloramphenicol (CLM) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino-acid analogues of CLM. The L-histidyl analogue binds to the ribosome with the affinity exceeding that of CLM by 10 fold. Several of the newly synthesized analogues were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CLM. However, the inhibitory properties of the semi-synthetic CLM-analogues did not correlate with their affinity and in general, the amino-acid analogues of CLM were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogues showed that CLM derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

The increase in multi-drug resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P- and E-site tRNAs bound, and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A-site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates, and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.