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Role of antibiotic ligand in nascent peptide-dependent ribosome stalling.

ABSTRACT: Specific nascent peptides in the ribosome exit tunnel can elicit translation arrest. Such ribosome stalling is used for regulation of expression of some bacterial and eukaryotic genes. The stalling is sensitive to additional cellular cues, most commonly the binding of specific small-molecular-weight cofactors to the ribosome. The role of cofactors in programmed translation arrest is unknown. By analyzing nascent peptide- and antibiotic-dependent ribosome stalling that controls inducible expression of antibiotic resistance genes in bacteria, we have found that the antibiotic is directly recognized as a part of the translation modulating signal. Even minute structural alterations preclude it from assisting in ribosome stalling, indicating the importance of precise molecular interactions of the drug with the ribosome. One of the sensors that monitor the structure of the antibiotic is the 23S rRNA residue C2610, whose mutation reduces the efficiency of nascent peptide- and antibiotic-dependent ribosome stalling. These findings establish a new paradigm of the role of the cofactor in programmed translation arrest in which a small molecule is recognized along with specific nascent peptide sequences as a composite structure that provokes arrest of translation. A similar mechanism could be used by the ribosome to sense a variety of cellular metabolites.

SUBMITTER: Vazquez-Laslop N

PROVIDER: S-EPMC3127923 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain.

Project description:The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson-Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.

| S-EPMC5499644 | biostudies-literature

Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel.

Project description:The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold.

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Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader peptide.

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Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

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Sequence requirements for ribosome stalling by the arginine attenuator peptide.

Project description:The 5' regions of eukaryotic mRNAs often contain upstream open reading frames (uORFs). The Neurospora crassa arg-2 uORF encodes the 24-residue arginine attenuator peptide (AAP). This regulatory uORF-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. The nascent AAP causes ribosomes to stall at the uORF stop codon in response to arginine, thus, blocking ribosomes from reaching the ARG-2 initiation codon. Here scanning mutagenesis with alanine and proline was performed to systematically determine which AAP residues were important for conferring regulation. Changing many of the most highly conserved residues (Asp-12, Tyr-13, Lys-14, and Trp-19) abolished regulatory function. The minimal functional domain of the AAP was determined by positioning AAP sequences internally within a large polypeptide. Pulse-chase analyses revealed that residues 9-20 of the AAP composed the minimal domain that was sufficient to confer regulatory function. An extensive analysis of predicted fungal AAPs revealed that the minimal functional domain of the N. crassa AAP corresponded closely to the region that was most highly conserved among the fungi. We also observed that the tripeptide RGD could function similarly to arginine in triggering AAP-mediated ribosome stalling. These studies provide a better understanding of the elements required for a nascent peptide and a small regulatory molecule to control translational processes.

| S-EPMC3003393 | biostudies-literature

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule.

Project description:The drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small number of proteins by an unknown mechanism. In structures of PF846-stalled human ribosome nascent chain complexes, PF846 binds in the ribosome exit tunnel in a eukaryotic-specific pocket formed by 28S ribosomal RNA, and alters the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state of translocation, in which the peptidyl-transfer RNA 3'-CCA end is improperly docked in the peptidyl transferase center. Selections of messenger RNAs from mRNA libraries using translation extracts reveal that PF846 can stall translation elongation, arrest termination or even enhance translation, depending on nascent chain sequence context. These results illuminate how a small molecule selectively targets translation by the human ribosome, and provides a foundation for developing small molecules that modulate the production of proteins of therapeutic interest.

| S-EPMC6919564 | biostudies-literature

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

Project description:A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ?-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ?-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

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The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling.

Project description:Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression of erm is activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the cotranscribed erm Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes the erm Shine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here, we show that nonsense mutations in the Staphylococcus aureus ErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence or presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of the ermBL-ermB transcript, and the macrolide appears to mitigate mRNA cleavage at a site immediately downstream of the ermBL SD sequence. The stabilizing effect of antibiotics on mRNA is not limited to ermBL-ermB; cationic antibiotics representing a ribosome-stalling inducer and a noninducer increase the half-life of specific transcripts. These data unveil a new layer of ermB regulation and imply that ErmBL translation or ribosome stalling serves as a "tuner" to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

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Computational analysis of nascent peptides that induce ribosome stalling and their proteomic distribution in <i>Saccharomyces cerevisiae</i>.

Project description:Interactions between the ribosomal exit tunnel and the nascent peptide can affect translation elongation rates. While previous studies have already demonstrated the feasibility of such interactions, little is known about the nature of the stalling peptide sequences and their distribution in the proteome. Here we ask which peptide sequences tend to occupy the tunnel of stalled ribosomes and how they are distributed in the proteome. Using computational analysis of ribosome profiling data from S. cerevisiae, we identified for the first time dozens of short stalling peptide sequences and studied their statistical properties. We found that short peptide sequences associated with ribosome stalling tend significantly to be either over- or underrepresented in the proteome. We then showed that the stalling interactions may occur at different positions along the length of the tunnel, prominently close to the P-site. Our findings throw light on the determinants of nascent peptide-mediated ribosome stalling during translation elongation and support the novel conjecture that mRNA translation affects the proteomic distribution of short peptide sequences.

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Nascent peptide assists the ribosome in recognizing chemically distinct small molecules.

Project description: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.

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