title: Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance technique.
abstract: BACKGROUND
  Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does.
  
  
  RESULTS
  The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G.
  
  
  CONCLUSIONS
  These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.
authors.affiliation: RRF Research Inc., Kannondai, Tsukuba, Ibaraki, 305-0856, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ishino
authors.forename: K
authors.initials: K
authors.lastname: Atarashi
authors.forename: S
authors.initials: S
authors.lastname: Uchiyama
authors.forename: T
authors.initials: T
authors.lastname: Yamami
authors.forename: Y
authors.initials: Y
authors.lastname: Saihara
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: H
authors.initials: H
authors.lastname: Hara
authors.forename: K
authors.initials: K
authors.lastname: Yokose
authors.forename: Y
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001120:Arginine / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D005672:Fusidic Acid / Q000494:pharmacology; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D011132:Polyribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D020871:RNA Stability / Q000187:drug effects / Q000235:genetics; D012335:RNA, Ribosomal / Q000378:metabolism; D012269:Ribosomal Proteins; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*; D013883:Thiostrepton / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D020653:Peptide Elongation Factor G; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; D006639:Histidine; D005672:Fusidic Acid; D001120:Arginine; D013883:Thiostrepton; D005998:Glycine