Functional mapping of ribosome-contact sites in the ribosome recycling factor: A structural view from a tRNA mimic

Fujiwara, Toshinobu; Ito, Koichi; Nakamura, Yoshikazu

doi:10.1017/s1355838201001704

Cited by 23 publications

(27 citation statements)

References 19 publications

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“…However, the gaps in our understanding of the mechanistic aspects of RRF function present a major limitation in the exploitation of this factor as a novel drug target. Mutational analyses, a crucial facet of structure-function studies, are important for a better understanding of the mechanism of action of RRF Fujiwara et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome

Rao

Varshney

2002

Microbiology

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Ribosome recycling factor (RRF), coded for by the frr locus, is involved in the disassembly of post-termination complexes and recycling of the ribosomes for a fresh round of initiation in bacteria and in eukaryotic organelles. In a crossspecies-complementation experiment, it was shown that the Thermus thermophilus RRF protein lacking five amino acids from its C-terminal end (∆C5TthRRF) but not the full-length protein (TthRRF) complemented Escherichia coli for its frr ts phenotype. It was also shown that the Mycobacterium tuberculosis RFF protein (MtuRRF) did not complement E. coli LJ14 for frr ts . However, simultaneous expression of elongation factor G (EFG) and RRF from M. tuberculosis resulted in complementation of E. coli LJ14. Here it is shown that unlike ∆C5TthRRF, an equivalent mutant of MtuRRF lacking six amino acids from its C-terminal end (∆C6MtuRRF) did not complement E. coli LJ14. Surprisingly, ∆C6MtuRRF failed to complement the strain even in the presence of homologous EFG (MtuEFG). The biochemical and biophysical characterization of these proteins suggested that the mutant RRF folded properly. However, ribosome-binding assays showed that the mutant protein was compromised in its binding to E. coli ribosomes. It is suggested that the conserved amino acids at the C-terminal end of the RRFs contribute to their residency on ribosomes and that the specific interactions between RRF and EFG are crucial in the disassembly of the termination complex.

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Section: Discussionmentioning

confidence: 99%

“…The creation of similar mutations in E. coli RRF (EcoRRF) showed that mutants which lacked up to seven residues from the Cterminal end were still functional. However, deletions that resulted in the loss of nine or more residues from the C-terminal end were defective in ribosome recycling (Fujiwara et al, 2001 ;Janosi et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome

Rao

Varshney

2002

Microbiology

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“…We suggest that this site is the same site where ec-RRF is moved to and released from the ribosomes during normal disassembly of the post-termination complexes. This hypothesis is supported by the fact that tt-RRF functions in E. coli that have defective ec-RRF (Fujiwara et al 2001). Therefore, binding of tt-RRF to ec-ribosomes described here is identical to that of ec-RRF.…”

Section: Wwwrnajournalorgmentioning

confidence: 94%

“…RRF is an essential protein in prokaryotes (Janosi et al 1994) and eukaryotes also (Rolland et al 1999;Teyssier et al 2003). The biological importance of RRF was further supported by isolation of a number of RRF mutants (Janosi et al 1998Fujiwara et al 1999Fujiwara et al , 2001) that have lethal effects. In the absence of RRF, ribosomes are not released from mRNA and start unscheduled translation downstream of the termination codon (Janosi et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins—insight into the mechanism of the ribosome recycling step

Raj

Kaji²,

Kaji³

2005

RNA

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Ribosome recycling factor (RRF), elongation factor-G (EF-G), and ribosomes from Thermus thermophilus (tt-) and Escherichia coli (ec-) were used to study the disassembly mechanism of post-termination ribosomal complexes by these factors. With tt-RRF, ec-EF-G can release bound-tRNA from ec-model post-termination complexes. However, tt-RRF is not released by ec-EF-G from ec-ribosomes. This complex with tt-RRF and ec-ribosomes after the tRNA release by ec-EF-G is regarded as an intermediate of the disassembly reaction. Not only tt-RRF, but also mRNA, cannot be released from ec-ribosomes by tt-RRF and ec-EF-G. These data suggest that the release of RRF from ribosomes is coupled or closely related to the release of mRNA during disassembly of post-termination complexes. With tt-ribosomes, ec-EF-G cannot release ribosome-bound ec-RRF even though they are from the same species, showing that proper interaction of ec-RRF and ec-EF-G does not occur on tt-ribosomes. On the other hand, in contrast to a published report, tt-EF-G functions with ec-RRF to disassemble ec-post-termination complexes. In support of this finding, tt-EF-G translocates peptidyl tRNA on ec-ribosomes and catalyzes ec-ribosome-dependent GTPase, showing that tt-EF-G has in vitro translocation activity with ec-ribosomes. Since tt-EF-G with ec-RRF can release tRNA from ec-post-termination complexes, the data are consistent with the hypothesis that the release of tRNA by RRF and EF-G from post-termination complexes is a result of a translocation-like activity of EF-G on RRF.

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“…RRFs whose structures have been determined to be a strict L-shape, were obtained from thermophilic bacteria and did not show polysome breakdown activity in vitro system containing EF-G and puromycin-treated polysome fraction from E. coli (11)(12)(13). RRFs that demonstrated activity in that system were only mesophilic bacteria, i.e.…”

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confidence: 99%

Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium

Nakano¹,

Yoshida²,

Uchiyama³

et al. 2003

Journal of Biological Chemistry

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X-ray and NMR analyses on ribosome recycling factors (RRFs) from thermophilic bacteria showed that they display a tRNA-like L-shaped conformation consisting of two domains. Since then, it has been accepted that domain I, consisting of a three-helix bundle, corresponds to the anticodon arm of tRNA and domain II and a ␤/␣/␤ sandwich structure, corresponds to the acceptor arm. In this study, we obtained a RRF from a mesophilic bacterium, Vibrio parahaemolyticus, by gene cloning and carried out an x-ray analysis on it at 2.2 Å resolution. This RRF was shown to be active in an in vitro assay system using Escherichia coli polysomes and elongation factor G (EF-G). In contrast, the above-mentioned RRFs from thermophilic bacteria were inactive in such a system. Analysis of the relative orientations between the two domains in the structures of various RRFs, including this RRF from mesophilic bacterium, revealed that domain II rotates about the long axis of the helix bundle of domain I. To elucidate the ribosome binding site of RRF, the peptide fragment (RRF-DI) corresponding to domain I of RRF was expressed and characterized. RRF-DI is bound to 70 S ribosome and the 50 S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30 S subunit. These findings caused us to reinvestigate the concept of the mimicry of RRF to tRNA and to propose a new model where domain I corresponds to the acceptor arm of tRNA and domain II corresponds to the anticodon arm. This is just the reverse of a model that is now widely accepted. However, the new model is in better agreement with published biological findings.

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Functional mapping of ribosome-contact sites in the ribosome recycling factor: A structural view from a tRNA mimic

Cited by 23 publications

References 19 publications

Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome

Characterization of Mycobacterium tuberculosis ribosome recycling factor (RRF) and a mutant lacking six amino acids from the C-terminal end reveals that the C-terminal residues are important for its occupancy on the ribosome

Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins—insight into the mechanism of the ribosome recycling step

Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium

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