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1.
Mol Cell ; 84(4): 715-726.e5, 2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38183984

ABSTRACT

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.


Subject(s)
Carboxylic Ester Hydrolases , Peptides , Ribosomes , Ribosomes/metabolism , Peptides/genetics , Bacteria/genetics , Quality Control , Protein Biosynthesis
2.
Cell ; 157(3): 529-31, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24766801

ABSTRACT

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.


Subject(s)
Escherichia coli/metabolism , Protein Biosynthesis
3.
Cell ; 151(3): 508-20, 2012 Oct 26.
Article in English | MEDLINE | ID: mdl-23101624

ABSTRACT

The polypeptide exit tunnel is an important functional compartment of the ribosome where the newly synthesized proteins are surveyed. The tunnel is the target of clinically important macrolide antibiotics. Macrolides plug the tunnel and are believed to stop production of all proteins. Contrary to this view, we show that drug-bound ribosomes can synthesize a distinct subset of cellular polypeptides. The structure of a protein defines its ability to thread through the antibiotic-obstructed tunnel. Synthesis of certain polypeptides that initially bypass translational arrest can be stopped at later stages of elongation while translation of some proteins goes to completion. Our findings reveal that small-molecule effectors can accentuate the discriminatory properties of the ribosomal exit tunnel and that macrolide antibiotics reshape the cellular proteome rather than block global protein synthesis.


Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Macrolides/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Ribosomes/drug effects , Amino Acid Sequence , Molecular Sequence Data , Peptide Chain Elongation, Translational , Ribosomes/metabolism
4.
Mol Cell ; 74(3): 481-493.e6, 2019 05 02.
Article in English | MEDLINE | ID: mdl-30904393

ABSTRACT

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.


Subject(s)
Genome, Bacterial/genetics , Peptide Chain Initiation, Translational , Proteome/genetics , Proteomics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Codon, Initiator/genetics , Diterpenes/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/drug effects , Genome, Bacterial/drug effects , RNA, Messenger/genetics , Ribosomes/drug effects , Ribosomes/genetics
5.
Nat Chem Biol ; 2024 Jul 22.
Article in English | MEDLINE | ID: mdl-39039256

ABSTRACT

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.

6.
Nucleic Acids Res ; 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38953159

ABSTRACT

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.

7.
J Biol Chem ; 300(3): 105780, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38395310

ABSTRACT

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.


Subject(s)
Escherichia coli , Protein Biosynthesis , Ribosomes , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Peptides/metabolism , Puromycin , Ribosomes/metabolism
8.
Nat Chem Biol ; 19(9): 1082-1090, 2023 09.
Article in English | MEDLINE | ID: mdl-36997647

ABSTRACT

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.


Subject(s)
Antimicrobial Peptides , Protein Biosynthesis , Animals , Escherichia coli/metabolism , Glycopeptides/chemistry , Drosophila/chemistry , Drosophila/metabolism
9.
Mol Cell ; 65(2): 207-219, 2017 Jan 19.
Article in English | MEDLINE | ID: mdl-28107647

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/biosynthesis , Cation Transport Proteins/biosynthesis , Copper/metabolism , Escherichia coli/enzymology , Frameshifting, Ribosomal , Molecular Chaperones/biosynthesis , Ribosomes/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Copper-Transporting ATPases , Escherichia coli/genetics , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Genotype , HEK293 Cells , Homeostasis , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutation , Nucleic Acid Conformation , Peptide Chain Termination, Translational , Phenotype , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transfection
11.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Article in English | MEDLINE | ID: mdl-35064089

ABSTRACT

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.


Subject(s)
Aminoglycosides/pharmacology , Binding Sites , Peptide Chain Initiation, Translational/drug effects , RNA, Messenger/genetics , Ribosomes/metabolism , Aminoglycosides/chemistry , Codon, Initiator , Molecular Structure , Open Reading Frames , Protein Binding , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Ribosomes/chemistry , Structure-Activity Relationship
12.
Nat Chem Biol ; 18(11): 1277-1286, 2022 11.
Article in English | MEDLINE | ID: mdl-36138139

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents , Ribosomes , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Ribosomes/metabolism , RNA, Transfer, Amino Acyl/genetics , RNA, Transfer, Amino Acyl/chemistry , RNA, Transfer, Amino Acyl/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , Amino Acid Sequence , Protein Biosynthesis
13.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Article in English | MEDLINE | ID: mdl-33674389

ABSTRACT

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.


Subject(s)
Antimicrobial Cationic Peptides , Escherichia coli , Peptide Chain Termination, Translational/drug effects , Protein Synthesis Inhibitors , Amino Acid Substitution , Animals , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Bees , Escherichia coli/genetics , Escherichia coli/metabolism , Mutation, Missense , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/pharmacology , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism
14.
Annu Rev Microbiol ; 72: 185-207, 2018 Sep 08.
Article in English | MEDLINE | ID: mdl-29906204

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Ribosomal/metabolism , Ribosomal Proteins/metabolism , Ribosomes/drug effects , Anti-Bacterial Agents/metabolism , Protein Binding , Protein Synthesis Inhibitors/metabolism
15.
Proc Natl Acad Sci U S A ; 117(4): 1971-1975, 2020 01 28.
Article in English | MEDLINE | ID: mdl-31932436

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/growth & development , Macrolides/pharmacology , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/metabolism , Ribosomes/metabolism , Anti-Bacterial Agents/chemistry , Base Pairing , Binding Sites , Escherichia coli/drug effects , Escherichia coli/genetics , Macrolides/chemistry , Nucleic Acid Conformation , Protein Biosynthesis , Protein Synthesis Inhibitors/pharmacology , RNA, Ribosomal, 23S/genetics
16.
Trends Biochem Sci ; 43(9): 668-684, 2018 09.
Article in English | MEDLINE | ID: mdl-30054232

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents , Macrolides , Protein Biosynthesis/drug effects , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Macrolides/pharmacokinetics , Macrolides/pharmacology
17.
J Bacteriol ; 204(1): e0029421, 2022 01 18.
Article in English | MEDLINE | ID: mdl-34339296

ABSTRACT

Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/metabolism , Open Reading Frames , Ribosomes , Bacteria/genetics , Codon , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/physiology , Peptide Chain Initiation, Translational , Peptide Chain Termination, Translational , RNA, Bacterial , RNA, Ribosomal
18.
Nat Chem Biol ; 16(3): 310-317, 2020 03.
Article in English | MEDLINE | ID: mdl-31844301

ABSTRACT

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.


Subject(s)
Chloramphenicol/pharmacology , Linezolid/pharmacology , Protein Biosynthesis/drug effects , Amino Acids/metabolism , Anti-Bacterial Agents/pharmacology , Binding Sites , Chloramphenicol/metabolism , Escherichia coli/metabolism , Fluorescence Resonance Energy Transfer/methods , Linezolid/metabolism , Peptides/metabolism , Protein Binding , RNA, Transfer/metabolism , Ribosomes/metabolism
19.
Mol Cell ; 56(3): 446-452, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25306253

ABSTRACT

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.


Subject(s)
Erythromycin/chemistry , Protein Biosynthesis , Protein Synthesis Inhibitors/chemistry , Ribosomes/chemistry , Bacterial Proteins/chemistry , Catalytic Domain , Cryoelectron Microscopy , Models, Molecular , Peptide Fragments/chemistry , Protein Binding , Protein Sorting Signals , Protein Structure, Quaternary , Ribosomes/physiology
20.
Mol Cell ; 56(4): 541-50, 2014 Nov 20.
Article in English | MEDLINE | ID: mdl-25306922

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/chemistry , Protein Synthesis Inhibitors/chemistry , RNA, Bacterial/chemistry , RNA, Ribosomal/chemistry , RNA, Transfer/chemistry , Amino Acid Motifs , Amino Acids, Diamino/chemistry , Base Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Protein Biosynthesis , RNA Stability , RNA, Messenger/chemistry , Ribosome Subunits, Large, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/chemistry , Thermus thermophilus
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