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Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1.

Hatin I, Fabret C, Rousset JP, Namy O - Nucleic Acids Res. (2009)

Bottom Line: We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity.Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1.Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud and IGM, CNRS, UMR 8621, Orsay, F 91405, France.

ABSTRACT
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

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Distribution of the mutations in the SUP45 gene and eRF1 protein. (A) Schematic representation of the SUP45 gene divided into three functional domains. The mutations identified in this study are represented alongside the gene by a gray circle for positions at which mutations were found once or twice, a red circle for mutations found three to five times, and a green circle for mutations found at least five times. All mutants studied are shown below, with the number of times they were found. Mutations found only once are indicated when they are the only mutation identified in the gene. (B) Schematic 3D representation of the eRF1 protein using 1DT9 pdb file. Amino- and carboxy-terminal extremities and each domain are indicated. Functional motifs (GGQ and NIKS) are also shown. Mutated residues are shown in stick representation in yellow and mutations are displayed in red or orange.
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Figure 1: Distribution of the mutations in the SUP45 gene and eRF1 protein. (A) Schematic representation of the SUP45 gene divided into three functional domains. The mutations identified in this study are represented alongside the gene by a gray circle for positions at which mutations were found once or twice, a red circle for mutations found three to five times, and a green circle for mutations found at least five times. All mutants studied are shown below, with the number of times they were found. Mutations found only once are indicated when they are the only mutation identified in the gene. (B) Schematic 3D representation of the eRF1 protein using 1DT9 pdb file. Amino- and carboxy-terminal extremities and each domain are indicated. Functional motifs (GGQ and NIKS) are also shown. Mutated residues are shown in stick representation in yellow and mutations are displayed in red or orange.

Mentions: Many studies have investigated eRF1 specificity for stop codons. However, most of these studies screened for eRF1 mutants with reduced termination efficiency (3,5,10–14). We developed a new approach based on the use of a weak stop codon to determine the role of the various eRF1 domains. Such a stop codon is easily suppressed by natural tRNAs, allowing a high rate of stop codon readthrough in a wild-type genetic background (15,16). We screened a random PCR mutagenesis library of the SUP45 gene to identify hyperactive mutants, using a method previously developed in our laboratory (17) (Figure 1 and Figure S1). Our study demonstrates that all the domains of the protein are involved in regulating termination efficiency. Moreover, we identified novel mutations in domains 2 and 3, which specifically improve termination at the UAA and UAG codons but have no effect on the UGA codon.Figure 1.


Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1.

Hatin I, Fabret C, Rousset JP, Namy O - Nucleic Acids Res. (2009)

Distribution of the mutations in the SUP45 gene and eRF1 protein. (A) Schematic representation of the SUP45 gene divided into three functional domains. The mutations identified in this study are represented alongside the gene by a gray circle for positions at which mutations were found once or twice, a red circle for mutations found three to five times, and a green circle for mutations found at least five times. All mutants studied are shown below, with the number of times they were found. Mutations found only once are indicated when they are the only mutation identified in the gene. (B) Schematic 3D representation of the eRF1 protein using 1DT9 pdb file. Amino- and carboxy-terminal extremities and each domain are indicated. Functional motifs (GGQ and NIKS) are also shown. Mutated residues are shown in stick representation in yellow and mutations are displayed in red or orange.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2665212&req=5

Figure 1: Distribution of the mutations in the SUP45 gene and eRF1 protein. (A) Schematic representation of the SUP45 gene divided into three functional domains. The mutations identified in this study are represented alongside the gene by a gray circle for positions at which mutations were found once or twice, a red circle for mutations found three to five times, and a green circle for mutations found at least five times. All mutants studied are shown below, with the number of times they were found. Mutations found only once are indicated when they are the only mutation identified in the gene. (B) Schematic 3D representation of the eRF1 protein using 1DT9 pdb file. Amino- and carboxy-terminal extremities and each domain are indicated. Functional motifs (GGQ and NIKS) are also shown. Mutated residues are shown in stick representation in yellow and mutations are displayed in red or orange.
Mentions: Many studies have investigated eRF1 specificity for stop codons. However, most of these studies screened for eRF1 mutants with reduced termination efficiency (3,5,10–14). We developed a new approach based on the use of a weak stop codon to determine the role of the various eRF1 domains. Such a stop codon is easily suppressed by natural tRNAs, allowing a high rate of stop codon readthrough in a wild-type genetic background (15,16). We screened a random PCR mutagenesis library of the SUP45 gene to identify hyperactive mutants, using a method previously developed in our laboratory (17) (Figure 1 and Figure S1). Our study demonstrates that all the domains of the protein are involved in regulating termination efficiency. Moreover, we identified novel mutations in domains 2 and 3, which specifically improve termination at the UAA and UAG codons but have no effect on the UGA codon.Figure 1.

Bottom Line: We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity.Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1.Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

View Article: PubMed Central - PubMed

Affiliation: Université Paris-Sud and IGM, CNRS, UMR 8621, Orsay, F 91405, France.

ABSTRACT
Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

Show MeSH