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Mick F Tuite - One of the best experts on this subject based on the ideXlab platform.
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Translation Termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism.
The EMBO journal, 1999Co-Authors: Simon S. Eaglestone, Brian S. Cox, Mick F TuiteAbstract:[PSI+] is a protein-based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion-like behaviour of the endogenous Sup35p protein release factor. [PSI+] strains exhibit a marked decrease in Translation Termination efficiency, which permits decoding of Translation Termination signals and, presumably, the production of abnormally extended polypeptides. We have examined whether the [PSI+]-induced expression of such an altered proteome might confer some selective growth advantage over [psi-] strains. Although otherwise isogenic [PSI+] and [psi-] strains show no difference in growth rates under normal laboratory conditions, we demonstrate that [PSI+] strains do exhibit enhanced tolerance to heat and chemical stress, compared with [psi-] strains. Moreover, we also show that the prion-like determinant [PSI+] is able to regulate Translation Termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of Translation Termination and a loss of the [PSI+] phenotype. We present a model to describe the prion-mediated regulation of Translation Termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi.
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The C-terminus of eRF1 defines a functionally important domain for Translation Termination in Saccharomyces cerevisiae.
Molecular microbiology, 1999Co-Authors: Lily Eurwilaichitr, Ian Stansfield, Fiona M. Graves, Mick F TuiteAbstract:Translation Termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates Termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for Translation Termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a Termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient Translation Termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.
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Translation Termination and its regulation in eukaryotes: recent insights provided by studies in yeast.
Biochemistry. Biokhimiia, 1999Co-Authors: Pierre M. Mugnier, Mick F TuiteAbstract:In protein synthesis, the arrival of one or other of the three stop codons in the ribosomal A-site triggers the binding of a release factor (RF) to the ribosome and subsequent polypeptide chain release. In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3. eRF1 is responsible for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function, although its precise role remains to be defined. Recent findings on Translation Termination and its regulation from studies in the yeast Saccharomyces cerevisiae are reviewed and the potential role of eRF3 is discussed.
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The influence of 5 ' codon context on Translation Termination in Saccharomyces cerevisiae
European journal of biochemistry, 1998Co-Authors: Salim Mottagui-tabar, Mick F Tuite, Leif A. IsakssonAbstract:Translation Termination in vivo was studied in the yeast Saccharomyces cerevisiae using a Translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on Termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient Termination. Thus, the C-terminal end of the nascent peptide influences Translation Termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of Termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in Termination efficiency. This suggests that the P-site tRNA is able to influence Termination at UGAC in yeast.
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a conditional lethal Translation Termination defect in a sup45 mutant of the yeast succhuromyces cerevisiue
FEBS Journal, 1997Co-Authors: Ian Stansfield, Vitaly V Kushnirov, Kerrie M Jones, Mick F TuiteAbstract:Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the Translational-Termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRFI), which has a stop-codon-dependent peptidyl-release activity. Using a conditional-lethal mutant allele of SUP45 (sup4.5-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for Translation Termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45–2 mutant strain exhibits a Termination defect when heated for short periods to greater than the non-permissive temperature (37°C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The Termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free Translation lysates from the sup45-2 strain do not show a defect in sense-codon Translation at the non-permissive temperature. These data demonstrate that yeast eRFl plays a role in Translation Termination and is functionally equivalent to its higher eukaryotic homologues.
Heike Krebber - One of the best experts on this subject based on the ideXlab platform.
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Translation Termination depends on the sequential ribosomal entry of eRF1 and eRF3.
Nucleic acids research, 2019Co-Authors: Christian Beißel, Bettina Neumann, Simon Uhse, Irene Hampe, Prajwal Karki, Heike KrebberAbstract:Translation Termination requires eRF1 and eRF3 for polypeptide- and tRNA-release on stop codons. Additionally, Dbp5/DDX19 and Rli1/ABCE1 are required; however, their function in this process is currently unknown. Using a combination of in vivo and in vitro experiments, we show that they regulate a stepwise assembly of the Termination complex. Rli1 and eRF3-GDP associate with the ribosome first. Subsequently, Dbp5-ATP delivers eRF1 to the stop codon and in this way prevents a premature access of eRF3. Dbp5 dissociates upon placing eRF1 through ATP-hydrolysis. This in turn enables eRF1 to contact eRF3, as the binding of Dbp5 and eRF3 to eRF1 is mutually exclusive. Defects in the Dbp5-guided eRF1 delivery lead to premature contact and premature dissociation of eRF1 and eRF3 from the ribosome and to subsequent stop codon readthrough. Thus, the stepwise Dbp5-controlled Termination complex assembly is essential for regular Translation Termination events. Our data furthermore suggest a possible role of Dbp5/DDX19 in alternative Translation Termination events, such as during stress response or in developmental processes, which classifies the helicase as a potential drug target for nonsense suppression therapy to treat cancer and neurodegenerative diseases.
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Translation Termination: new factors and insights.
RNA biology, 2010Co-Authors: Claudia Baierlein, Heike KrebberAbstract:In eukaryotes, Translation Termination requires two eukaryotic release factors, eRF1 and eRF3. eRF1 is required for recognition of the stop codon and eRF3 supports the polypeptide chain release in a GTP dependent manner. Recently, several new players in Translation Termination have been identified. The DEAD-box RNA helicase Dbp5 has been shown to support eRF1 in stop codon recognition, possibly by proper placement of the release factor on the Termination codon. Upon its dissociation from eRF1, Dbp5 allows the entry of the second Termination factor eRF3 into the complex. Further, the Dbp5 interacting protein Gle1 and its co-factor inositol hexakisphosphate (IP6) have been shown to participate in the Termination process. Dbp5 and Gle1 are well known for their function in mRNA export from the nucleus to the cytoplasm. Most interestingly, also the ATP binding cassette (ABC) protein Rli1, which requires the mitochondrial and cytosolic Fe/S protein biogenesis machineries for its assembly, has recently been show...
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The iron-sulphur protein RNase L inhibitor functions in Translation Termination.
EMBO reports, 2010Co-Authors: Sohail Khoshnevis, Thomas Gross, Claudia Baierlein, Carmen Rotte, Ralf Ficner, Heike KrebberAbstract:The iron–sulphur (Fe–S)-containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and Translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in Translation Termination. Through co-immunoprecipitation experiments, we show that Rli1 interacts physically with the Translation Termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35-21 or sup45-2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other Termination factors. By contrast, RLI1 overexpression partly suppresses the read-through defects in sup45-2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in Translation Termination; an Fe–S cluster mutant of RLI1 cannot suppress the read-through defects of sup45-2.
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The DEAD-Box RNA Helicase Dbp5 Functions in Translation Termination
Science, 2007Co-Authors: Thomas Gross, Anja Siepmann, Merle Windgassen, John J. Scarcelli, Charles N. Cole, Matthias Seedorf, Dorothee Sturm, Heike KrebberAbstract:In eukaryotes, Termination of messenger RNA (mRNA) Translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in Translation Termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into Termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.
David M. Bedwell - One of the best experts on this subject based on the ideXlab platform.
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Tpa1p is part of an mRNP complex that influences Translation Termination, mRNA deadenylation, and mRNA turnover in Saccharomyces cerevisiae.
Molecular and cellular biology, 2006Co-Authors: Kim M. Keeling, Joe Salas-marco, Lev Osherovich, David M. BedwellAbstract:In this report, we show that the Saccharomyces cerevisiae protein Tpa1p (for Termination and polyadenylation) influences Translation Termination efficiency, mRNA poly(A) tail length, and mRNA stability. Tpa1p is encoded by the previously uncharacterized open reading frame YER049W. Yeast strains carrying a deletion of the TPA1 gene (tpa1) exhibited increased readthrough of stop codons, and coimmunoprecipitation assays revealed that Tpa1p interacts with the Translation Termination factors eRF1 and eRF3. In addition, the tpa1 mutation led to a 1.5- to 2-fold increase in the half-lives of mRNAs degraded by the general 533 pathway or the 335 nonstop decay pathway. In contrast, this mutation did not have any affect on the nonsense-mediated mRNA decay pathway. Examination of mRNA poly(A) tail length revealed that poly(A) tails are longer than normal in a tpa1 strain. Consistent with a potential role in regulating poly(A) tail length, Tpa1p was also found to coimmunoprecipitate with the yeast poly(A) binding protein Pab1p. These results suggest that Tpa1p is a component of a messenger ribonucleoprotein complex bound to the 3 untranslated region of mRNAs that affects Translation Termination, deadenylation, and mRNA decay.
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GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination
Molecular and Cellular Biology, 2004Co-Authors: Joe Salas-marco, David M. BedwellAbstract:Translation Termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the Translation Termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of Translation Termination at some Termination signals but not others. As much as a 17-fold decrease in the Termination efficiency was observed at some tetranucleotide Termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other Termination signals. To determine whether this stop signal-dependent decrease in the efficiency of Translation Termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of Translation Termination rather than a decrease at a subset of Termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of Translation Termination at any Termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of Translation Termination signals by eRF1 to efficient polypeptide chain release.
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The Efficiency of Translation Termination is Determined by a Synergistic Interplay Between Upstream and Downstream Sequences inSaccharomyces cerevisiae
Journal of molecular biology, 1995Co-Authors: Brian Bonetti, James J. Moon, David M. BedwellAbstract:Abstract In a recent study we found that the efficiency of Translation Termination could be decreased several hundred fold by altering the local sequence context surrounding stop codons in the yeastSaccharomyces cerevisiae. Suppression of Termination was shown to be mediated by near-cognate tRNA mispairing with the Termination codon. We have now examined in greater detail how the local sequence context affects the efficiency of Translation Termination in this organism. Our results indicate that the sequence immediately upstream of the Termination codon plays a significant role in determining the efficiency of Translation Termination. An extended Termination sequence (containing the stop codon and the following three nucleotides) was also found to be a major determinant of Termination efficiency, with effects attributable to the fourth nucleotide being largely independent of the Termination codon. For the UGA and UAA stop codons, the influence of the fourth position on Termination efficiency (from most efficient to least efficient Termination) was found to be G>U,A>C, while for the UAG codon it was U,A>C>G. These sequence-specific effects on the efficiency of Translation Termination suggest that polypeptide chain release factor (or another molecule that may play a role in Translation Termination, such as rRNA) recognizes an extended Termination sequence in yeast. A previous study found a statistically significant bias toward certain tetranucleotide sequences (containing the stop codon and the first distal nucleotide) in several organisms. We found that tetranucleotide sequences most frequently used in yeast are among the most efficient at mediating Translation Termination, while rare tetranucleotide sequences mediate much less efficient Termination. Taken together, our results indicate that upstream and downstream components of an extended sequence context act synergistically to determine the overall efficiency of Translation Termination in yeast.
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Premature Translation Termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family.
The Journal of biological chemistry, 1994Co-Authors: K Fearon, V Mcclendon, B Bonetti, David M. BedwellAbstract:Abstract The requirements for efficient Translation Termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of Translation Termination, premature Termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature Translation Termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature Termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of Translation Termination. Amino acid sequence analysis of the full-length Translation products produced by read through of an amber codon demonstrated that Termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.
Andrei A. Korostelev - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of premature Translation Termination on a sense codon.
Journal of Biological Chemistry, 2018Co-Authors: Egor Svidritskiy, Garbriel Demo, Andrei A. KorostelevAbstract:Accurate Translation Termination by release factors (RFs) is critical for the integrity of cellular proteomes. Premature Termination on sense codons, for example, results in truncated proteins, whose accumulation could be detrimental to the cell. Nevertheless, some sense codons are prone to triggering premature Termination, but the structural basis for this is unclear. To investigate premature Termination, we determined a cryo-EM structure of the Escherichia coli 70S ribosome bound with RF1 in response to a UAU (Tyr) sense codon. The structure reveals that RF1 recognizes a UAU codon similarly to a UAG stop codon, suggesting that sense codons induce premature Termination because they structurally mimic a stop codon. Hydrophobic interaction between the nucleobase of U3 (the third position of the UAU codon) and conserved Ile-196 in RF1 is important for misreading the UAU codon. Analyses of RNA binding in ribonucleoprotein complexes or by amino acids reveal that Ile-U packing is a frequent protein-RNA-binding motif with key functional implications. We discuss parallels with eukaryotic Translation Termination by the release factor eRF1.
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Mechanism of Inhibition of Translation Termination by Blasticidin S.
Journal of molecular biology, 2018Co-Authors: Egor Svidritskiy, Andrei A. KorostelevAbstract:Understanding the mechanisms of inhibitors of Translation Termination may inform development of new antibacterials and therapeutics for premature Termination diseases. We report the crystal structure of the potent Termination inhibitor blasticidin S bound to the ribosomal 70S•release factor 1 (RF1) Termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in Termination accuracy.
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Conformational Control of Translation Termination on the 70S Ribosome.
Structure, 2018Co-Authors: Egor Svidritskiy, Andrei A. KorostelevAbstract:Summary Translation Termination ensures proper lengths of cellular proteins. During Termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate Translation Termination. However, structural studies of ribosome Termination complexes have only captured RFs in a conformation that is consistent with the catalytically active state. Here, we employ a hyper-accurate RF1 variant to obtain crystal structures of 70S Termination complexes that suggest a structural pathway for RF1 activation. We trapped RF1 conformations with the catalytic domain outside of the peptidyl-transferase center, while the codon-recognition domain binds the stop codon. Stop-codon recognition induces 30S decoding-center rearrangements that precede accommodation of the catalytic domain. The separation of codon recognition from the opening of the catalytic domain suggests how rearrangements in RF1 and in the ribosomal decoding center coordinate stop-codon recognition with peptide release, ensuring accurate Translation Termination.
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Conformational control of Translation Termination on the 70S ribosome
2017Co-Authors: Egor Svidritskiy, Andrei A. KorostelevAbstract:Translation Termination ensures proper lengths of cellular proteins. During Termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate Translation Termination. If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome Termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S Termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codon-recognition domain is fully engaged in stop-codon recognition in the decoding center. RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate Translation Termination.
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Structural Basis for Translation Termination on a Pseudouridylated Stop Codon.
Journal of molecular biology, 2016Co-Authors: Egor Svidritskiy, Rohini Madireddy, Andrei A. KorostelevAbstract:Pseudouridylation of messenger RNA emerges as an abundant modification involved in gene expression regulation. Pseudouridylation of stop codons in eukaryotic and bacterial cells results in stop-codon read through. The structural mechanism of this phenomenon is not known. Here we present a 3.1-Å crystal structure of Escherichia coli release factor 1 (RF1) bound to the 70S ribosome in response to the ΨAA codon. The structure reveals that recognition of a modified stop codon does not differ from that of a canonical stop codon. Our in vitro biochemical results support this finding by yielding nearly identical rates for peptide release from E. coli ribosomes programmed with pseudouridylated and canonical stop codons. The crystal structure also brings insight into E. coli RF1-specific interactions and suggests involvement of L27 in bacterial Translation Termination. Our results are consistent with a mechanism in which read through of a pseudouridylated stop codon in bacteria results from increased decoding by near-cognate tRNAs (miscoding) rather than from decreased efficiency of Termination.
Elena Alkalaeva - One of the best experts on this subject based on the ideXlab platform.
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Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination
2020Co-Authors: Alexey Shuvalov, Ekaterina Shuvalova, Tatiana Egorova, Elizaveta Sokolova, Biziaev N, Evmenov K, Elena AlkalaevaAbstract:The Nsp1 protein of SARS-CoV-2 regulates the Translation of host and viral mRNAs in cells. Nsp1 inhibits host Translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-Termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-Termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in Translation Termination. Using a cell-free Translation system and reconstituted in vitro Translation system, we show that Nsp1 stimulates Translation Termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in Translation Termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in Translation Termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.
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CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.
Biomolecules, 2020Co-Authors: Kseniya A. Lashkevich, Elena Alkalaeva, Valeriya I Shlyk, Artem S Kushchenko, Vadim N. Gladyshev, Sergey E. DmitrievAbstract:Translation Termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of Translation Termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro Translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before Translation Termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the Translation dynamics of these two reporters allows visualization of a delay corresponding to the Translation Termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the Translation Termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on Termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic Translation system, mostly by affecting elongation, and that an excess of eRF1 Termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.
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The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes
Molekuliarnaia biologiia, 2020Co-Authors: Elizaveta Sokolova, Alexey Shuvalov, Tatiana Egorova, Petr Vlasov, Elena AlkalaevaAbstract:Translation Termination is a finishing step of protein biosynthesis. The significant role in this process belongs not only to protein factors of Translation Termination but also to the nearest nucleotide environment of stop codons. There are numerous descriptions of stop codons readthrough, which is due to specific nucleotide sequences behind them. However, represented data are segmental and don't explain the mechanism of the nucleotide context influence on Translation Termination. It is well known that stop codon UAA usage is preferential for A/T-rich genes, and UAG, UGA-for G/C-rich genes, which is related to an expression level of these genes. We investigated the connection between a frequency of nucleotides occurrence in 3' area of stop codons in the human genome and their influence on Translation Termination efficiency. We found that 3' context motif, which is cognate to the sequence of a stop codon, stimulates Translation Termination. At the same time, the nucleotide composition of 3' sequence that differs from stop codon, decreases Translation Termination efficiency.
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Polyadenylate-binding protein–interacting proteins PAIP1 and PAIP2 affect Translation Termination
The Journal of biological chemistry, 2019Co-Authors: A. V. Ivanov, Ekaterina Shuvalova, Tatiana Egorova, Alexey Shuvalov, Elizaveta Sokolova, Nikita Bizyaev, Ivan N. Shatsky, Ilya M. Terenin, Elena AlkalaevaAbstract:Polyadenylate-binding protein (PABP) stimulates Translation Termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its Translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on Translation Termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented Translation Termination at the premature Termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on Translation Termination in vitro. However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated Translation Termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-Termination complex. These results indicated that PAIP1 and PAIP2 participate in Translation Termination and are important regulators of readthrough at the premature Termination codon.
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PABP enhances release factor recruitment and stop codon recognition during Translation Termination.
Nucleic acids research, 2016Co-Authors: Alexandr Ivanov, Boris Eliseev, Alexey Shuvalov, Elizaveta Sokolova, Lahari Yeramala, Christiane Schaffitzel, Tatyana Mikhailova, Denis Susorov, Elena AlkalaevaAbstract:Poly(A)-binding protein (PABP) is a major component of the messenger RNA-protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as Translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate Translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro Translation system, we show that PABP directly stimulates Translation Termination. PABP increases the efficiency of Translation Termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in Translation Termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-Termination complexes (preTCs) significantly increases the efficiency of peptidyl-tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1-eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in Translation Termination.
