Molecular Mechanisms Of Archaeal And Bacterial Ribosome Translocation And Inhibition Of Translocation By Viomycin

The ribosome is a molecular machine that is responsible for protein translation.Ribosome translocation is the coupled movements of tRNA-mRNA from the A and P sites to the P and E sites,which is a fundamental step in protein translation and widely conserved in bacteria,eukaryotes and archaea.In this study,we investigated the mechanisms of translocation in archaea and bacteria.In archaea,We studied a specie of Crenarchaeota named Sulfolobus acidocaldarius(Sac)and solved multiple structures of translocation intermediates by cry o-EM with resolution ranging from 2.75.7 ?.The structures provided important insights into mechanisms of translocation and translation under extreme environment in archaea.In the subunit structures,we observed unstable conformations of H68 and h44 of rRNA,which may interfere with subunit association.The subunit structures provided models for 12 rRNA expansion segments and 3 novel r-proteins,providing structural insights into our understanding of archaeal growth under extreme conditions.The structure of the 50S-aRFl complex showed the unique domain orientation of aRF1,which possibly explaining P-site tRNA release after translation termination.The Sac 70S complexes were captured in 7 distinct steps of the tRNA translocation reaction,confirming conserved structural features during archaeal ribosome translocation.In aEF2-70S ribosome complexes,3D classification of cryo-EM data based on 30S head domain identified two new translocation intermediate states with 30S head domain tilted 5°-6° enabling its disengagement from the translocated tRNA and its release post-translocation,explaining the molecular mechanism of how ribosomes release tRNAs that have moved during the late phase of translocation.Conformational changes of aEF2 during ribosome binding and switching from three different states were described.Our structures illustrate details of crenarchaeal ribosome architecture and provide new insights into ribosome translocation.The ribosomes are targets for many clinical antibiotics,these drugs can interfer with the protein translation,thereby inhibit bacterial growth.We study viomycin,an antibiotic that has been used to fight tuberculosis infections and inhibit the ribosome translocation in the pathogenic bacteria.Viomycin has been shown to stabilize the ribosome in a state of inter-subunit rotation,resembling the hybrid-state intermediate of translocation.Viomycin primarily was also found to inhibit ribosomal subunit dissociation.The mechanism by which viomycin stabilizes hybrid state and inhibits ribosome dissociation remains not fully understood.To address the unsolved questions,we have determined cryo-EM and X-ray crystal structures of E.coli 70S ribosome complexes trapped in a rotated state by viomycin.The 3.8-? cryo-EM structure revealed a ribosome trapped in the hybrid state with 8.6° inter-subunit rotation and 5.3°rotation of the 30S subunit head domain,bearing a single P/E tRNA.We identified five different binding sites for viomycin,four of which have not been described previously.To resolve the details of their binding interactions,we solved the 3.1-? crystal structure of a viomycin-bound ribosome complex,revealing that all five viomycins bind to ribosomal RNA.One of these(Vio1)corresponds to the single viomycin that was previously identified in a complex with a classical-state ribosome.The Vio3,Vio4,and Vio5 were clustered at inter-subunit bridges,consistent with the ability of viomycin to inhibit subunit dissociation.We propose that one or more of these same three viomycins induce inter-subunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23 S rRNA,blocking conformational changes associated with molecular movements that are required for translocation.The newly discovered binding sites provide structural basis for structure-based drug development.