Abstract
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.
Highlights
Protein synthesis occurs on ribosomes in three basic steps of initiation [1, 2], elongation [3,4,5], and termination [6]
Elongation factor G (EF-G) optimally works at an approximate 1:1 ratio with the complexes. This is consistent with earlier studies that had shown that the optimal rate of ribosome recycling occurs when EF-G and ribosome recycling factor (RRF) are present at a 1:1 ratio [13], and it demonstrates that EF-G works stoichiometrically with RRF
We previously proposed that P/E site tRNA would be released by RRF and EF-G [17]
Summary
Buffers—The buffers used were as follows: buffer A, 20 mM Tris-HCl (pH 7.5), 100 mM NH4Cl, 10 mM Mg(OAc) mM DTT; buffer B, 20 mM Tris-HCl (pH 7.5), 10 mM Mg(OAc) 500 mM NH4Cl, 2 mM DTT; buffer C, 20 mM Tris-HCl (pH 7.5), 50 mM NH4Cl, 10 mM Mg(OAc) mM DTT; buffer D, 10 mM Tris-HCl (pH 7.5), 10 mM MgSO4, 50 mM NH4Cl, 0.5 mM DTT; buffer E, 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT; buffer F, 5 mM potassium phosphate buffer (pH 7), 1 mM DTT; buffer G, 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 10 mM Mg(OAc); buffer H, 14 mM Tris-HCl (pH 7.4), 12 mM Mg(OAc) mM NH4Cl, 0.3 mM DTT; buffer I, 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 7 mM Mg(OAc) M urea; buffer J, 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 7 mM Mg(OAc) 300 mM imidazole. In the experiments where the binding of RRF to preformed complexes of ribosome, EF-G, and fusidic acid were studied, preformed complexes were prepared by incubating 100 pmol of ribosomes, 250 pmol of EF-G, 1 mM GTP, and 1 mM fusidic acid in 40 l of buffer G for 10 min. In both cases, free EF-G was removed, bound EF-G was quantified, and the complexes were reacted with RRF as described above. No RRF was detected in the absence of polysome. tRNA release from the polysome was measured by amino-acylation of nitrocellulose (0.45 m) filtrate as described [16]
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