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J. Biol. Chem., Vol. 278, Issue 48, 48041-48050, November 28, 2003

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Release of Ribosome-bound Ribosome Recycling Factor by Elongation Factor G^*

Michael C. Kiel, V. Samuel Raj, Hideko Kaji, and Akira Kaji¶

From the Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Philadelphia, Pennsylvania 19107

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.

Received for publication, May 8, 2003 , and in revised form, August 11, 2003.

^* This work was supported in part by National Institutes of Health Grant GH60429 (to A. K.) and the Nippon Paint Research Fund (to H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Microbiology, School of Medicine, University of Pennsylvania, Rm. 203B Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-898-8828; Fax: 215-573-2221; E-mail: kaji{at}mail.med.upenn.edu.

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