Dual functions of ribosome recycling factor in protein biosynthesis: Disassembling the termination complex and preventing translational errors

Janosi, Laszlo; Ricker, Robert D.; Kaji, A.

doi:10.1016/s0300-9084(97)86718-1

Cited by 63 publications

(61 citation statements)

References 39 publications

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“…After the centrifugation of the homogenate at 300,000 ϫ g for 3 h, the supernatant was applied onto a 5-ml Hi-Trap Q column (Amersham Biosciences) equilibrated with buffer A. The flowthrough fraction containing RRF was dialyzed against buffer B (20 mM sodium acetate, pH 5.0, 10 mM NH 4 Cl, and 6 mM 2-mercaptoethanol) and applied onto a 5-ml Hi-Trap SP column (Amersham Biosciences) equilibrated with buffer B. The eluted fractions containing RRF were concentrated by Centricon YM-10 (Millipore), and the concentrate was further purified by a Superdex 75 pg column (Amersham Biosciences) equilibrated with buffer A containing 300 mM sodium acetate.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction mixture (150 l) contained 10 mM Tris-HCl at pH 7.4, 8.2 mM MgCl 2 , 80 mM NH 4 Cl, 1 mM DTT, 0.16 mM GTP, 0.05 mM puromycin, 1.3 A 260 units of polysomes, 1.6 M EF-G, and various amounts of RRF. The mixture was incubated at 30°C for 20 min, followed by separation of the mixture into polysome and monosomes with 5-45% sucrose density gradient ultracentrifugation (250,000 ϫ g, 45 min at 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…Binding 4 Cl, 1 mM DTT, and 0.3 mM EDTA) at 30°C for 10 min. The mixture was applied onto Microcon YM-100 (Millipore) and centrifuged for 5 min at 3,000 ϫ g to trap the ribosome-bound RRF-DI.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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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“…34,35 This ribosome recycling provides the bacterial cell with 30S and 50S ribosomal subunits competent for initiating the synthesis of new protein molecules. 36 Thus, defective ribosome recycling should decrease the productivity of protein synthesis.…”

Section: Is the Ribosome Recycling Step Defective In The L25 Mutant?mentioning

confidence: 99%

Importance of the 5 S rRNA-binding Ribosomal Proteins for Cell Viability and Translation in Escherichia coli

Korepanov

Gongadze

Garber

et al. 2007

Journal of Molecular Biology

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SummaryA specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.

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“…In vivo, following termination and in the absence of RRF, prokaryote 70S ribosomes can remain on the mRNA and continue to ' slide ' downstream, and eventually reinitiate protein synthesis at any codon between 17 and 45 nt downstream in a frame-independent manner (Janosi et al, 1998). In addition, RRF acting with EF-G can also reduce frameshift errors in vitro, and reduce missense translation error during peptide elongation (Janosi et al, 1996), although the mechanism of this activity is not well understood. The recently solved crystal structure of RRF from Thermotoga maritima has revealed that this protein, like eRF1, is a tRNA mimic, although a much more exact mimic than is eRF1 ( Fig.…”

Section: Post-termination Ribosomal Fatesmentioning

confidence: 99%

Endless possibilities: translation termination and stop codon recognition

et al. 2001

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OverviewThe process of protein synthesis can be divided into three main phases : initiation, during which the ribosomal subunits join the mRNA and locate the AUG initiator codon ; elongation, during which sense codons are decoded and the bulk of the polypeptide is made ; and termination, during which a stop codon directs the release of the completed polypeptide from the ribosome. Whereas the general principles of sense codon decoding by transfer RNAs are well established, a clear picture of translation termination and stop codon recognition has hitherto been lacking. Recently however, the use of optimized, complex, reconstituted in vitro termination reactions to identify the roles of key termination factors, and the solution of tertiary structures of termination factors, has allowed a reappraisal of termination factor structure and function. This review will describe these recent advances, many of which have resulted from studies of termination in micro-organisms, and in the light of the new information, discuss models for the mechanism of termination and recognition of the stop codon. While the mechanism of peptide chain termination is normally effective, stop codons are not always recognized efficiently, and during the translation of certain viral RNAs can be suppressed or read through, resulting in the expression of additional coding information. How stop codons are reassigned to encode ' sense ' at a given frequency in some RNAs will be reviewed in the context of current understanding of termination and stop codon recognition mechanisms. The translation termination apparatus in eukaryotes and prokaryotesDuring translation termination, a stop codon located in the ribosomal A-site is recognized by a release factor or release factor complex, which binds the ribosome and triggers release of the nascent peptide. In eukaryotes, translation is terminated by a heterodimer consisting of two proteins, release factors eRF1 and eRF3, which interact in vivo (Frolova et al., 1994 ;Zhouravleva et al., 1995 ;Stansfield et al., 1995). eRF1 recognizes all three stop codons and triggers peptidyl-tRNA hydrolysis by the ribosome, releasing the nascent peptide (Frolova et al., 1994 ; Drugeon et al., 1997). Eukaryote termination efficiency is enhanced by the GTPase release factor eRF3, the second component of the heterodimer eRF complex. In response to a stop codon in the ribosomal A-site, formation of a quaternary complex comprising the ribosome, eRF1, GTP and eRF3 triggers GTP hydrolysis and enhances the rate of peptidyl release (Zhouravleva et al., 1995 ; Frolova et al., 1994 ; Fig. 1). In yeast, eRF1 and eRF3 are encoded by essential genes (SUP35 and SUP45, respectively), mutations in which produce nonsense suppression phenotypes (Stansfield & Tuite, 1994).In contrast to eukaryotes, the role of stop codon recognition during translation termination in eubacteria is divided between two so-called class 1 release factors, RF1 and RF2, which in Escherichia coli are encoded by the essential prfA and prfB genes, respectively (Scolni...

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Dual functions of ribosome recycling factor in protein biosynthesis: Disassembling the termination complex and preventing translational errors

Cited by 63 publications

References 39 publications

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

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

Importance of the 5 S rRNA-binding Ribosomal Proteins for Cell Viability and Translation in Escherichia coli

Endless possibilities: translation termination and stop codon recognition

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