Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).
- MeSH
- ABC transportéry chemie genetika metabolismus MeSH
- alosterická regulace účinky léků genetika MeSH
- antibakteriální látky chemie farmakologie MeSH
- Bacillus subtilis enzymologie genetika MeSH
- bakteriální léková rezistence * MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- ribozomy chemie genetika metabolismus MeSH
- RNA transferová chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.
- MeSH
- elektronová kryomikroskopie MeSH
- Escherichia coli genetika metabolismus MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- kationické antimikrobiální peptidy farmakologie MeSH
- konformace proteinů MeSH
- peptidy - faktory ukončení metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- proteosyntéza účinky léků MeSH
- ribozomální proteiny metabolismus MeSH
- ribozomy metabolismus MeSH
- RNA transferová metabolismus MeSH
- simulace molekulového dockingu MeSH
- terminace translace peptidového řetězce MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- velké podjednotky ribozomu bakteriální metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.
- MeSH
- chloroplasty chemie MeSH
- elektronová kryomikroskopie MeSH
- malé podjednotky ribozomu eukaryotické chemie MeSH
- molekulární modely MeSH
- proteiny chloroplastové chemie metabolismus MeSH
- ribozomální proteiny chemie metabolismus MeSH
- RNA ribozomální chemie MeSH
- Spinacia oleracea chemie MeSH
- stabilita RNA MeSH
- vazebná místa MeSH
- velké podjednotky ribozomu eukaryotické chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.
- MeSH
- elektronová kryomikroskopie MeSH
- elongační faktory chemie genetika metabolismus ultrastruktura MeSH
- Escherichia coli genetika metabolismus MeSH
- iniciační faktory chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- messenger RNA chemie genetika metabolismus MeSH
- mutace MeSH
- peptidy chemie metabolismus MeSH
- proteiny vázající RNA chemie metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus ultrastruktura MeSH
- proteosyntéza MeSH
- ribozomy chemie metabolismus ultrastruktura MeSH
- RNA transferová chemie genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
