Release of ribosome-bound ribosome recycling factor by elongation factor G.
Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.
Kiel
Michael C
MC
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Raj
V Samuel
VS
Kaji
Hideko
H
Kaji
Akira
A
D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005672:Fusidic Acid / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D020653:Peptide Elongation Factor G / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*; D013883:Thiostrepton / Q000494:pharmacology; D013997:Time Factors; D014756:Viomycin / Q000494:pharmacology
D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D013883:Thiostrepton; D014756:Viomycin