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Comprehensive genetic selection revealed essential bases in the peptidyl-transferase center.

ABSTRACT: During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.

SUBMITTER: Sato NS

PROVIDER: S-EPMC1592644 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center.

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Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

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2016-09-08 | GSE86536 | GEO

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Project description:We combine genetic perturbation with cryo-electron microscopy (cryo-EM) to establish the mechanism of PTC maturation during human mitoribosome biogenesis. Cryo-EM structures of large mitoribosomal subunit (mtLSU) assembly intermediates purified from GTPBP6-deficient cells reveal that GTPBP5 acts in concert with the methyltransferase domain of the NSUN4-MTERF4 complex to facilitate PTC folding. Addition of recombinant GTPBP6 to these assembly intermediates provides the structural basis for subsequent GTPBP6-mediated late PTC maturation and suggests a sequential order of mtLSU biogenesis. Finally, cryo-EM analysis of 55S mitoribosomes treated with GTPBP6 explains how this protein can adopt a dual role in ribosome biogenesis and recycling. Taken together, these results provide the molecular basis for PTC maturation and establish a hierarchical model for late mitoribosome biogenesis.

2021-05-20 | PXD023502 | Pride

SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center.

Project description:As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA-nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNA(Pro) in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.

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Project description:The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N5 position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control. It has been suggested that the GGQ motif arose independently through convergent evolution among otherwise unrelated proteins that catalyze peptide release. The requirement for this tripeptide in the highly conserved peptidyl transferase center suggests that the conformation reported here is likely shared during termination of protein synthesis in all domains of life.

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Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center.

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

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