Intersubunit movement is required for ribosomal translocation.
Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The "ratchet model," based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes.
Horan
Lucas H
LH
Department of Molecular, Cell, and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
Noller
Harry F
HF
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D020653:Peptide Elongation Factor G; D012333:RNA, Messenger