The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Lyubov A Ryabova - One of the best experts on this subject based on the ideXlab platform.
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viral factor tav recruits tor s6k1 signalling to activate Reinitiation after long orf translation
The EMBO Journal, 2011Co-Authors: Mikhail Schepetilnikov, Kappei Kobayashi, Angele Geldreich, Carole Caranta, Christophe Robaglia, Mario Keller, Lyubov A RyabovaAbstract:The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral Reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes Reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor Reinitiation-supporting protein (RISP) to regenerate Reinitiation-competent ribosomal complexes. Here, we show that TAV function in Reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation Reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and Reinitiation. Thus, activation of Reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of Reinitiation-competent ribosomal complexes.
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Viral factor TAV recruits TOR/S6K1 signalling to activate Reinitiation after long ORF translation.
The EMBO Journal, 2011Co-Authors: Mikhail Schepetilnikov, Kappei Kobayashi, Angele Geldreich, Carole Caranta, Christophe Robaglia, Mario Keller, Lyubov A RyabovaAbstract:The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral Reinitiation factor transactivator–viroplasmin (TAV) exceptionally promotes Reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor Reinitiation-supporting protein (RISP) to regenerate Reinitiation-competent ribosomal complexes. Here, we show that TAV function in Reinitiation depends on physical association with TOR, with TAV–TOR binding being critical for both translation Reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP—a novel and specific target of TOR/S6K1—in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and Reinitiation. Thus, activation of Reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of Reinitiation-competent ribosomal complexes.
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translation Reinitiation and leaky scanning in plant viruses
Virus Research, 2006Co-Authors: Lyubov A Ryabova, Mikhail M Pooggin, Thomas HohnAbstract:While translation of mRNAs in eukaryotic cells in general follows strict rules, viruses infecting these cells break those rules in various ways. Viruses are under high selection pressure to compete with the host, to economize genome size, and to accommodate signals for replication, virus assembly, etc., on their RNAs as well as using them for translation. The cornucopia of extraordinary translation strategies, such as leaky scanning, internal initiation of translation, ribosome shunt, and virus-controlled Reinitiation of translation, evolved by viruses continues to surprise and inform our understanding of general translation mechanisms. While internal initiation is treated in another section of this issue, we concentrate on leaky scanning, shunt and Reinitiation, with emphasis on plant pararetroviruses.
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eucaryotic initiation factor 4b controls eif3 mediated ribosomal entry of viral Reinitiation factor
The EMBO Journal, 2004Co-Authors: Hyunsook Park, Karen S Browning, Thomas Hohn, Lyubov A RyabovaAbstract:The cauliflower mosaic virus Reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the Reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated Reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.
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a plant viral Reinitiation factor interacts with the host translational machinery
Cell, 2001Co-Authors: Hyunsook Park, Axel Himmelbach, Karen S Browning, Thomas Hohn, Lyubov A RyabovaAbstract:Abstract The cauliflower mosaic virus transactivator, TAV, controls translation Reinitiation of major open reading frames on polycistronic RNA. We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3 and the 60S ribosomal subunit. Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated Reinitiation activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by Reinitiation, overcoming the normal cell barriers to this process.
Giorgio Dieci - One of the best experts on this subject based on the ideXlab platform.
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Investigating transcription Reinitiation through in vitro approaches
Transcription, 2014Co-Authors: Giorgio Dieci, Beatrice Fermi, Maria Cristina BosioAbstract:By influencing the number of RNA molecules repeatedly synthesized from the same gene, the control of transcription Reinitiation has the potential to shape the transcriptome. Transcription Reinitiation mechanisms have been mainly addressed in vitro, through approaches based on both crude and reconstituted systems. These studies support the notion that transcription Reinitiation and its regulation rely on dedicated networks of molecular interactions within transcription machineries. At the same time, comparison with in vivo transcription rates suggests that additional mechanisms, factors and conditions must exist in the nucleus, whose biochemical elucidation is a fascinating challenge for future in vitro transcription studies.
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transcription Reinitiation by rna polymerase iii
Biochimica et Biophysica Acta, 2013Co-Authors: Giorgio Dieci, Maria Cristina Bosio, Beatrice Fermi, Roberto FerrariAbstract:Abstract The retention of transcription proteins at an actively transcribed gene contributes to maintenance of the active transcriptional state and increases the rate of subsequent transcription cycles relative to the initial cycle. This process, called transcription Reinitiation, generates the abundant RNAs in living cells. The persistence of stable pReinitiation intermediates on activated genes representing at least a subset of basal transcription components has long been recognized as a shared feature of RNA polymerase (Pol) I, II and III-dependent transcription in eukaryotes. Studies of the Pol III transcription machinery and its target genes in eukaryotic genomes over the last fifteen years, has uncovered multiple details on transcription Reinitiation. In addition to the basal transcription factors that recruit the polymerase, Pol III itself can be retained on the same gene through multiple transcription cycles by a facilitated recycling pathway. The molecular bases for facilitated recycling are progressively being revealed with advances in structural and functional studies. At the same time, progress in our understanding of Pol III transcriptional regulation in response to different environmental cues points to the specific mechanism of Pol III Reinitiation as a key target of signaling pathway regulation of cell growth. This article is part of a Special Issue entitled: Transcription by Odd Pols.
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The transcription Reinitiation properties of RNA polymerase III in the absence of transcription factors
Cellular & Molecular Biology Letters, 2007Co-Authors: Roberto Ferrari, Giorgio DieciAbstract:Transcription Reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and Reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated Reinitiation during subsequent cycles is compromised.
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distinct roles of transcription factors tfiiib and tfiiic in rna polymerase iii transcription Reinitiation
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Roberto Ferrari, Claudio Rivetti, Joel Acker, Giorgio DieciAbstract:Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable pReinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, Reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated Reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short (≈100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, Reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high Reinitiation rate. Transcription Reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III Reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.
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Transcription Reinitiation properties of bacteriophage T7 RNA polymerase
Biochemical and Biophysical Research Communications, 2004Co-Authors: Roberto Ferrari, Claudio Rivetti, Giorgio DieciAbstract:Abstract We have analyzed the kinetics of transcription initiation and Reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP). We used a short transcription unit with T7-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones. When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1 min. Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10 min; after that time, Reinitiation practically ceased. When the polymerase was in large excess over template DNA, however, Reinitiation proceeded linearly for longer times, at a rate of 1 cycle/min. Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations.
Thomas Hohn - One of the best experts on this subject based on the ideXlab platform.
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translation Reinitiation and leaky scanning in plant viruses
Virus Research, 2006Co-Authors: Lyubov A Ryabova, Mikhail M Pooggin, Thomas HohnAbstract:While translation of mRNAs in eukaryotic cells in general follows strict rules, viruses infecting these cells break those rules in various ways. Viruses are under high selection pressure to compete with the host, to economize genome size, and to accommodate signals for replication, virus assembly, etc., on their RNAs as well as using them for translation. The cornucopia of extraordinary translation strategies, such as leaky scanning, internal initiation of translation, ribosome shunt, and virus-controlled Reinitiation of translation, evolved by viruses continues to surprise and inform our understanding of general translation mechanisms. While internal initiation is treated in another section of this issue, we concentrate on leaky scanning, shunt and Reinitiation, with emphasis on plant pararetroviruses.
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eucaryotic initiation factor 4b controls eif3 mediated ribosomal entry of viral Reinitiation factor
The EMBO Journal, 2004Co-Authors: Hyunsook Park, Karen S Browning, Thomas Hohn, Lyubov A RyabovaAbstract:The cauliflower mosaic virus Reinitiation factor TAV interacts with host translation initiation factor 3 (eIF3) and the 60S ribosomal subunit to accomplish translation of polycistronic mRNAs. Interaction between TAV and eIF3g is critical for the Reinitiation process. Here, we show that eIF4B can preclude formation of the TAV/eIF3 complex via competition with TAV for eIF3g binding; indeed, the eIF4B- and TAV-binding sites on eIF3g overlap. Our data indicate that eIF4B interferes with TAV/eIF3/40S ribosome complex formation during the first initiation event. Consequently, overexpression of TAV in plant protoplasts affects only second initiation events. Transient overexpression of eIF4B in plant protoplasts specifically inhibits TAV-mediated Reinitiation of a second ORF. These data suggest that TAV enters the host translation machinery at the eIF4B removal step to stabilize eIF3 on the translating ribosome, thereby allowing translation of polycistronic viral RNA.
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a plant viral Reinitiation factor interacts with the host translational machinery
Cell, 2001Co-Authors: Hyunsook Park, Axel Himmelbach, Karen S Browning, Thomas Hohn, Lyubov A RyabovaAbstract:Abstract The cauliflower mosaic virus transactivator, TAV, controls translation Reinitiation of major open reading frames on polycistronic RNA. We show here that TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 in vitro and in vivo. TAV physically interacts with eIF3 and the 60S ribosomal subunit. Two proteins mediating these interactions were identified: eIF3g and 60S ribosomal protein L24. Transient expression of eIF3g and L24 in plant protoplasts strongly affects TAV-mediated Reinitiation activity. We demonstrate that TAV/eIF3/40S and eIF3/TAV/60S ternary complexes form in vitro, and propose that TAV mediates efficient recruitment of eIF3 to polysomes, allowing translation of polycistronic mRNAs by Reinitiation, overcoming the normal cell barriers to this process.
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ribosome shunting in the cauliflower mosaic virus 35s rna leader is a special case of Reinitiation of translation functioning in plant and animal systems
Genes & Development, 2000Co-Authors: Lyubov A Ryabova, Thomas HohnAbstract:The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning‐Reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses Reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5*-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5*-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of Reinitiation.
Roberto Ferrari - One of the best experts on this subject based on the ideXlab platform.
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transcription Reinitiation by rna polymerase iii
Biochimica et Biophysica Acta, 2013Co-Authors: Giorgio Dieci, Maria Cristina Bosio, Beatrice Fermi, Roberto FerrariAbstract:Abstract The retention of transcription proteins at an actively transcribed gene contributes to maintenance of the active transcriptional state and increases the rate of subsequent transcription cycles relative to the initial cycle. This process, called transcription Reinitiation, generates the abundant RNAs in living cells. The persistence of stable pReinitiation intermediates on activated genes representing at least a subset of basal transcription components has long been recognized as a shared feature of RNA polymerase (Pol) I, II and III-dependent transcription in eukaryotes. Studies of the Pol III transcription machinery and its target genes in eukaryotic genomes over the last fifteen years, has uncovered multiple details on transcription Reinitiation. In addition to the basal transcription factors that recruit the polymerase, Pol III itself can be retained on the same gene through multiple transcription cycles by a facilitated recycling pathway. The molecular bases for facilitated recycling are progressively being revealed with advances in structural and functional studies. At the same time, progress in our understanding of Pol III transcriptional regulation in response to different environmental cues points to the specific mechanism of Pol III Reinitiation as a key target of signaling pathway regulation of cell growth. This article is part of a Special Issue entitled: Transcription by Odd Pols.
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The transcription Reinitiation properties of RNA polymerase III in the absence of transcription factors
Cellular & Molecular Biology Letters, 2007Co-Authors: Roberto Ferrari, Giorgio DieciAbstract:Transcription Reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and Reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated Reinitiation during subsequent cycles is compromised.
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distinct roles of transcription factors tfiiib and tfiiic in rna polymerase iii transcription Reinitiation
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Roberto Ferrari, Claudio Rivetti, Joel Acker, Giorgio DieciAbstract:Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable pReinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, Reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated Reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short (≈100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, Reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high Reinitiation rate. Transcription Reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III Reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.
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Transcription Reinitiation properties of bacteriophage T7 RNA polymerase
Biochemical and Biophysical Research Communications, 2004Co-Authors: Roberto Ferrari, Claudio Rivetti, Giorgio DieciAbstract:Abstract We have analyzed the kinetics of transcription initiation and Reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP). We used a short transcription unit with T7-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones. When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1 min. Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10 min; after that time, Reinitiation practically ceased. When the polymerase was in large excess over template DNA, however, Reinitiation proceeded linearly for longer times, at a rate of 1 cycle/min. Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations.
Gregor Meyers - One of the best experts on this subject based on the ideXlab platform.
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feline calicivirus can tolerate gross changes of its minor capsid protein expression levels induced by changing translation Reinitiation frequency or use of a separate vp2 coding mrna
PLOS ONE, 2014Co-Authors: Christine Luttermann, Gregor MeyersAbstract:Caliciviruses use Reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune Reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.
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two alternative ways of start site selection in human norovirus Reinitiation of translation
Journal of Biological Chemistry, 2014Co-Authors: Christine Luttermann, Gregor MeyersAbstract:The calicivirus minor capsid protein VP2 is expressed via termination/Reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for Reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for Reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of Reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that Reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although Reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.
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the importance of inter and intramolecular base pairing for translation Reinitiation on a eukaryotic bicistronic mrna
Genes & Development, 2009Co-Authors: Christine Luttermann, Gregor MeyersAbstract:Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/Reinitiation mechanism, which depends on an upstream sequence element of ∼70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two short sequence motifs within the TURBS were found to be essential for Reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation Reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/Reinitiation in eukaryotes.
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characterization of the sequence element directing translation Reinitiation in rna of the calicivirus rabbit hemorrhagic disease virus
Journal of Virology, 2007Co-Authors: Gregor MeyersAbstract:The calicivirus minor capsid protein VP2 is expressed via Reinitiation of protein synthesis after termination of translation of the preceding VP1 gene. A sequence element of about 80 nucleotides denoted "termination upstream ribosomal binding site" (TURBS) (25) is crucial for Reinitiation. Deletion mapping in the TURBS of a rabbit calicivirus identified two short sequence motifs that were crucial for VP2 expression. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18S rRNA. Single-residue exchanges in this motif severely impaired Reinitiation when they affected the putative rRNA binding, whereas an exchange preserving complementarity had only a minor effect. Single exchanges in motif 2 were rather well tolerated, but the introduction of double exchanges almost blocked VP2 expression. In contrast, the deletion analyses showed that the RNA between the two motifs is of minor importance. The distance between motif 2 and the start site was,found to be important, since deletions of increasing length in this sequence or upstream positioning of the start codon reduced VP2 expression stepwise to low levels, whereas multiple-nucleotide exchanges in this region were tolerated. The low flexibility of the arrangement of TURBS motif 2 and the start codon stand in marked contrast to the requirements with regard to the location of the stop codon of the preceding VP1 gene, which could be moved far downstream with continuous reduction, but without loss, of VP2 translation. The sequence mapping resulted in a refined model of the Reinitiation mechanism leading to VP2 expression
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a bipartite sequence motif induces translation Reinitiation in feline calicivirus rna
Journal of Biological Chemistry, 2007Co-Authors: Christine Luttermann, Gregor MeyersAbstract:The mechanism leading to Reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/Reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/Reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.
