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Warren P. Tate - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic translational termination efficiency is influenced by the 3 nucleotides within the ribosomal mrna channel
Nucleic Acids Research, 2018Co-Authors: Andrew G Cridge, Caillan Crowemcauliffe, Suneeth F Mathew, Warren P. TateAbstract:When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding Release Factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the Release Factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.
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mtrf1a is a human mitochondrial translation Release Factor decoding the major termination codons uaa and uag
Molecular Cell, 2007Co-Authors: Hamid Reza Soleimanpourlichaei, Warren P. Tate, Inge Kuhl, Mauricette Gaisne, Joao F Passos, Mateusz Wydro, Joanna Rorbach, Richard J Temperley, Nathalie Bonnefoy, Robert N LightowlersAbstract:Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key Factors have yet to be characterized—for example, those required for translation termination. All other systems have two classes of Release Factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I Factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I Release Factors. Although we could not confirm the function of this Factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.
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indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the Factor recycling function of escherichia coli Release Factor rf3
The EMBO Journal, 1999Co-Authors: Debbiejane G Crawford, Koichi Ito, Yoshikazu Nakamura, Warren P. TateAbstract:Prokaryotic Release Factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding Release Factors RF1 and RF2. The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide Release by displacing the Factors from the ribosome. In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength. The efficiency of decoding strong stop signals (e.g. UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected. However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect. The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF. These findings can be interpreted according to current views of RF3 as a recycling Factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.
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Translational termination in Escherichia coli: Three bases following the stop codon crosslink to Release Factor 2 and affect the decoding efficiency of UGA-containing signals
Nucleic Acids Research, 1998Co-Authors: Elizabeth S Poole, Sally A Mannering, Louise L. Major, Warren P. TateAbstract:The observations that the Escherichia coli Release Factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop signals, stimulated us to determine whether there was a more extended termination signal for RF2 recognition. Analysis of the 34 contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element. Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10). Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2. A strong selection bias for particular bases in the +4 to +6 positions of certain E.coli UGANNN termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo termination signal strength. These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence termination signal decoding efficiency.
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escherichia coli Release Factor 3 resolving the paradox of a typical g protein structure and atypical function with guanine nucleotides
RNA, 1998Co-Authors: Herman Jan Pel, Koichi Ito, Yoshikazu Nakamura, John G Moffat, Warren P. TateAbstract:Escherichia coli Release Factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding Release Factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding Release Factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding Release Factor while it enhances the efficiency of the termination reaction.
Lev L Kisselev - One of the best experts on this subject based on the ideXlab platform.
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the glutamine residue of the conserved ggq motif in saccharomyces cerevisiae Release Factor erf1 is methylated by the product of the ydr140w gene
Journal of Biological Chemistry, 2005Co-Authors: Valerie Heurguehamard, Stephanie Champ, Tatiana Merkoulovarainon, Liliana Mora, Lev L Kisselev, Richard H. BuckinghamAbstract:Abstract Polypeptide Release Factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to Release Factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N5-methyl-Gln by the S-adenosyl l-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the Release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae Release Factor eRF1, the first time that N5-methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial Release Factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl l-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1·eRF3·GTP.
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invariant amino acids essential for decoding function of polypeptide Release Factor erf1
Nucleic Acids Research, 2005Co-Authors: P M Kolosov, Ludmila Frolova, Alim S Seitnebi, V I Dubovaya, Artem V Kononenko, Nina Yu Oparina, Just Justesen, Alexandr Efimov, Lev L KisselevAbstract:In eukaryotic ribosome, the N domain of polypeptide Release Factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its Release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.
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termination of translation interplay of mrna rrnas and Release Factors
The EMBO Journal, 2003Co-Authors: Lev L Kisselev, Mans Ehrenberg, Ludmila FrolovaAbstract:Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain Release Factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.
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substitutions of the glutamine residue in the ubiquitous ggq tripeptide in human erf1 do not entirely abolish the Release Factor activity
Molecular Biology, 2000Co-Authors: Seit A Nebi, Liliya Frolova, N V Ivanova, A B Poltaraus, Lev L KisselevAbstract:Translation termination in eukaryotes takes place on the ribosome and requires the presence of a stop codon (any of the three) and of the eRF1 protein--a class-1 polypeptide chain Release Factor--at the ribosomal A site (reviewed in [1, 2]). Primary [3] and 3D [4] structures of human eRF1 have been established, making it possible to study the functioning of this protein. Regardless of the species, all class1 Release Factors share a common tripeptide GGQ, absolutely conserved in Eukarya, Archaea, and Prokarya [5]. This tripeptide occupies the minidomain "tip" at one of the extremities of the Y-shaped human eRF1 molecule [4].
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translation termination in eukaryotes polypeptide Release Factor erf1 is composed of functionally and structurally distinct domains
RNA, 2000Co-Authors: Ludmila Frolova, Tatyana I Merkulova, Lev L KisselevAbstract:Class-1 polypeptide chain Release Factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another Factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.
Ludmila Frolova - One of the best experts on this subject based on the ideXlab platform.
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two step model of stop codon recognition by eukaryotic Release Factor erf1
Nucleic Acids Research, 2013Co-Authors: Polina N Kryuchkova, A V Grishi, Oris Eliseev, Anna S. Karyagina, Ludmila Frolova, Elena AlkalaevaAbstract:Release Factor eRF1 plays a key role in the termination of protein synthesis in eukaryotes. The eRF1 consists of three domains (N, M and C) that perform unique roles in termination. Previous studies of eRF1 point mutants and standard/variant code eRF1 chimeras unequivocally demonstrated a direct involvement of the highly conserved N-domain motifs (NIKS, YxCxxxF and GTx) in stop codon recognition. In the current study, we extend this work by investigating the role of the 41 invariant and conserved N-domain residues in stop codon decoding by human eRF1. Using a combination of the conservative and non-conservative amino acid substitutions, we measured the functional activity of >80 mutant eRF1s in an in vitro reconstituted eukaryotic translation system and selected 15 amino acid residues essential for recognition of different stop codon nucleotides. Furthermore, toe-print analyses provide evidence of a conformational rearrangement of ribosomal complexes that occurs during binding of eRF1 to messenger RNA and reflects stop codon decoding activity of eRF1. Based on our experimental data and molecular modelling of the N-domain at the ribosomal A site, we propose a two-step model of stop codon decoding in the eukaryotic ribosome.
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invariant amino acids essential for decoding function of polypeptide Release Factor erf1
Nucleic Acids Research, 2005Co-Authors: P M Kolosov, Ludmila Frolova, Alim S Seitnebi, V I Dubovaya, Artem V Kononenko, Nina Yu Oparina, Just Justesen, Alexandr Efimov, Lev L KisselevAbstract:In eukaryotic ribosome, the N domain of polypeptide Release Factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its Release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.
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stop codons and ugg promote efficient binding of the polypeptide Release Factor erf1 to the ribosomal a site
Journal of Molecular Biology, 2003Co-Authors: Laurent Chavatte, Ludmila Frolova, Philippe Laugâa, L L Kisselev, Alain FavreAbstract:Abstract To investigate the codon dependence of human eRF1 binding to the mRNA–ribosome complex, we examined the formation of photocrosslinks between ribosomal components and mRNAs bearing a photoactivable 4-thiouridine probe in the first position of the codon located in the A site. Addition of eRF1 to the phased mRNA–ribosome complexes triggers a codon-dependent quenching of crosslink formation. The concentration of eRF1 triggering half quenching ranges from low for the three stop codons, to intermediate for s4UGG and high for other near-cognate triplets. A theoretical analysis of the photochemical processes occurring in a two-state bimolecular model raises a number of stringent conditions, fulfilled by the system studied here, and shows that in any case sound KD values can be extracted if the ratio mT/KD≪1 (mT is total concentration of mRNA added). Considering the KD values obtained for the stop, s4UGG and sense codons (≈0.06 μM, 0.45 μM and 2.3 μM, respectively) and our previous finding that only the stop and s4UGG codons are able to promote formation of an eRF1–mRNA crosslink, implying a role for the NIKS loop at the tip of the N domain, we propose a two-step model for eRF1 binding to the A site: a codon-independent bimolecular step is followed by an isomerisation step observed solely with stop and s4UGG codons. Full recognition of the stop codons by the N domain of eRF1 triggers a rearrangement of bound eRF1 from an open to a closed conformation, allowing the universally conserved GGQ loop at the tip of the M domain to come into close proximity of the peptidyl transferase center of the ribosome. UGG is expected to behave as a cryptic stop codon, which, owing to imperfect eRF1-codon recognition, does not allow full reorientation of the M domain of eRF1. As far as the physical steps of eRF1 binding to the ribosome are considered, they appear to closely mimic the behaviour of the tRNA/EF-Tu/GTP complex, but clearly eRF1 is endowed with a greater conformational flexibility than tRNA.
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termination of translation interplay of mrna rrnas and Release Factors
The EMBO Journal, 2003Co-Authors: Lev L Kisselev, Mans Ehrenberg, Ludmila FrolovaAbstract:Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain Release Factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.
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stop codon recognition in ciliates euplotes Release Factor does not respond to reassigned uga codon
EMBO Reports, 2001Co-Authors: Stephanie Kervesti, Ludmila Frolova, Lev Kisselev, Olivie JeanjeaAbstract:In eukaryotes, the polypeptide Release Factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.
Mans Ehrenberg - One of the best experts on this subject based on the ideXlab platform.
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the structural basis for Release Factor activation during translation termination revealed by time resolved cryogenic electron microscopy
Nature Communications, 2019Co-Authors: Gabriele Indrisiunaite, Mans Ehrenberg, Bo Chen, Sandip Kaledhonkar, Binita Shah, Ming Sun, Robert A Grassucci, Joachim FrankAbstract:When the ribosome encounters a stop codon, it recruits a Release Factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 A away. Surprisingly, free RF2 is compact, with only 20 A between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 A resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms. Translation termination is under strong selection pressure for high speed and accuracy. Here the authors provide a 3D view of the dynamics of a translating bacterial ribosome as it recruits a class-1 Release Factor (RF1 or RF2) upon encountering a stop codon, and propose a structure-based kinetic model for the early steps in bacterial translation termination.
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the structural basis for Release Factor activation during translation termination revealed by time resolved cryogenic electron microscopy
bioRxiv, 2018Co-Authors: Gabriele Indrisiunaite, Mans Ehrenberg, Bo Chen, Sandip Kaledhonkar, Binita Shah, Ming Sun, Robert A Grassucci, Joachim FrankAbstract:Abstract When the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 Release Factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 A away from the DC. The finding that free RF2 is compact with only 20 A between its codon reading and GGQ motifs came therefore as a surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM9,10,11,12,13 to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 A resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.
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On the pH Dependence of Class-1 RF-Dependent Termination of mRNA Translation
Journal of molecular biology, 2015Co-Authors: Gabriele Indrisiunaite, Valérie Heurgué-hamard, Michael Y. Pavlov, Mans EhrenbergAbstract:We have studied the pH dependence of the rate of termination of bacterial protein synthesis catalyzed by a class-1 Release Factor (RF1 or RF2). We used a classical quench-flow technique and a newly ...
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the role of ribosomal protein l11 in class i Release Factor mediated translation termination and translational accuracy
Journal of Biological Chemistry, 2006Co-Authors: Lamine Bouakaz, Mans Ehrenberg, Elli Bouakaz, Emanuel J Murgola, Suparna SanyalAbstract:It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide Release by, in particular, the class I Release Factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (ΔL11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4-6-fold reductions in termination efficiency (kcat/Km) of RF1, but not of RF2, on ΔL11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide Release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.
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termination of translation interplay of mrna rrnas and Release Factors
The EMBO Journal, 2003Co-Authors: Lev L Kisselev, Mans Ehrenberg, Ludmila FrolovaAbstract:Termination of translation in eukaryotes has focused recently on functional anatomy of polypeptide chain Release Factor, eRF1, by using a variety of different approaches. The tight correlation between the domain structure and different functions of eRF1 has been revealed. Independently, the role of prokaryotic RF1/2 in GTPase activity of RF3 has been deciphered, as well as RF3 function itself.
Yoshikazu Nakamura - One of the best experts on this subject based on the ideXlab platform.
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isolation and expression of two genes encoding eukaryotic Release Factor 1 from paramecium tetraurelia
Journal of Eukaryotic Microbiology, 2002Co-Authors: Stephanie Kervestin, Yoshikazu Nakamura, Eric Meyer, Olivier Garnier, Andrey L Karamyshev, Koichi Ito, Olivier JeanjeanAbstract:Abstract Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic Release Factor one (eRF1), a Factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1-a and eRF1-b, coding for eRF1 in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRF1 sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRF1 of P. tetraurelia exhibits significant differences in the N-termina...
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the mechanism of tryptophan induction of tryptophanase operon expression tryptophan inhibits Release Factor mediated cleavage of tnac peptidyl trnapro
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Feng Gong, Yoshikazu Nakamura, Charles YanofskyAbstract:Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNAPro) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNAPro accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of Release Factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNAPro and Release of TnaC from the ribosome-mRNA complex. Release Factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNAPro was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNAPro inhibits both TnaC peptidyl-tRNAPro hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho Factor binding and subsequent transcription termination.
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indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the Factor recycling function of escherichia coli Release Factor rf3
The EMBO Journal, 1999Co-Authors: Debbiejane G Crawford, Koichi Ito, Yoshikazu Nakamura, Warren P. TateAbstract:Prokaryotic Release Factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding Release Factors RF1 and RF2. The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide Release by displacing the Factors from the ribosome. In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength. The efficiency of decoding strong stop signals (e.g. UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected. However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect. The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF. These findings can be interpreted according to current views of RF3 as a recycling Factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.
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single amino acid substitution in prokaryote polypeptide Release Factor 2 permits it to terminate translation at all three stop codons
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Yoshikazu NakamuraAbstract:Abstract Prokaryotic translational Release Factors, RF1 and RF2, catalyze polypeptide Release at UAG/UAA and UGA/UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1/RF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent Release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic Release Factors share the same anticodon moiety and that only one omnipotent Release Factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent Factor.
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escherichia coli Release Factor 3 resolving the paradox of a typical g protein structure and atypical function with guanine nucleotides
RNA, 1998Co-Authors: Herman Jan Pel, Koichi Ito, Yoshikazu Nakamura, John G Moffat, Warren P. TateAbstract:Escherichia coli Release Factor 3 (RF3) is a G protein involved in the termination of protein synthesis that stimulates the activity of the stop signal decoding Release Factors RF1 and RF2. Paradoxically for a G protein, both GDP and GTP have been reported to modulate negatively the activity of nucleotide-free RF3 in vitro. Using a direct ribosome binding assay, we found that RF3xGDPCP, a GTP analogue form of RF3, has a 10-fold higher affinity for ribosomes than the GDP form of the protein, and that RF3xGDPCP binds to the ribosome efficiently in the absence of the decoding Release Factors. These effects show that RF3 binds to the ribosome as a classical translational G protein, and suggest that the paradoxical inhibitory effect of GTP on RF3 activity in vitro is most likely due to untimely and unproductive ribosome-mediated GTP hydrolysis. Nucleotide-free RF3 has an intermediate activity and its binding to the ribosome exhibits positive cooperativity with RF2. This cooperativity is absent, however, in the presence of GDPCP. The observed activities of nucleotide-free RF3 suggest that it mimics a transition state of RF3 in which the protein interacts with the decoding Release Factor while it enhances the efficiency of the termination reaction.