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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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Hyperactive eRF1 mutants can complement the deletion of the SUP45 gene. The FS1ΔS strain was transformed with a plasmid carrying sup45 mutants and plated on selective media for this plasmid. Rescue plasmid carrying wild-type SUP45 gene was shuffled by plating the colonies on media containing 5FOA. The growing cells were transferred to a selective liquid culture until they reached 1 OD600 and were serially diluted. Plates were incubated either at 30 °C or 37 °C to test for thermosensitivity. Ts denotes the MT557/3b strain carrying a thermosensitive sup45 allele; E104K, E360G; Q76R, E360G; N58K, E360G; are double mutants created by directed mutagenesis, combining mutations identified independently during the screen within the same molecule.
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Figure 2: Hyperactive eRF1 mutants can complement the deletion of the SUP45 gene. The FS1ΔS strain was transformed with a plasmid carrying sup45 mutants and plated on selective media for this plasmid. Rescue plasmid carrying wild-type SUP45 gene was shuffled by plating the colonies on media containing 5FOA. The growing cells were transferred to a selective liquid culture until they reached 1 OD600 and were serially diluted. Plates were incubated either at 30 °C or 37 °C to test for thermosensitivity. Ts denotes the MT557/3b strain carrying a thermosensitive sup45 allele; E104K, E360G; Q76R, E360G; N58K, E360G; are double mutants created by directed mutagenesis, combining mutations identified independently during the screen within the same molecule.

Mentions: We carried out our genetic screen in the presence of the wild-type SUP45 gene. Translational elongation mutants with an elevated level of translational accuracy interfere with cell viability (20). Termination mutants with an elevated level of accuracy may thus severely affect yeast growth rate. To determine the potential effects of these mutant proteins on growth, we constructed a yeast strain deleted for SUP45 (FS1ΔS) rescued by a URA3 plasmid bearing wild-type SUP45. We selected a subset of mutants carrying only a single mutation, or a combination of these single mutations, for this analysis. The FS1ΔS strain was then transformed with the mutant constructs and the wild-type SUP45 gene was shuffled. Our results (Figure 2) clearly demonstrated that all the mutant cells were viable at 30°C. However, all three double mutants were thermosensitive.Figure 2.


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)

Hyperactive eRF1 mutants can complement the deletion of the SUP45 gene. The FS1ΔS strain was transformed with a plasmid carrying sup45 mutants and plated on selective media for this plasmid. Rescue plasmid carrying wild-type SUP45 gene was shuffled by plating the colonies on media containing 5FOA. The growing cells were transferred to a selective liquid culture until they reached 1 OD600 and were serially diluted. Plates were incubated either at 30 °C or 37 °C to test for thermosensitivity. Ts denotes the MT557/3b strain carrying a thermosensitive sup45 allele; E104K, E360G; Q76R, E360G; N58K, E360G; are double mutants created by directed mutagenesis, combining mutations identified independently during the screen within the same molecule.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665212&req=5

Figure 2: Hyperactive eRF1 mutants can complement the deletion of the SUP45 gene. The FS1ΔS strain was transformed with a plasmid carrying sup45 mutants and plated on selective media for this plasmid. Rescue plasmid carrying wild-type SUP45 gene was shuffled by plating the colonies on media containing 5FOA. The growing cells were transferred to a selective liquid culture until they reached 1 OD600 and were serially diluted. Plates were incubated either at 30 °C or 37 °C to test for thermosensitivity. Ts denotes the MT557/3b strain carrying a thermosensitive sup45 allele; E104K, E360G; Q76R, E360G; N58K, E360G; are double mutants created by directed mutagenesis, combining mutations identified independently during the screen within the same molecule.
Mentions: We carried out our genetic screen in the presence of the wild-type SUP45 gene. Translational elongation mutants with an elevated level of translational accuracy interfere with cell viability (20). Termination mutants with an elevated level of accuracy may thus severely affect yeast growth rate. To determine the potential effects of these mutant proteins on growth, we constructed a yeast strain deleted for SUP45 (FS1ΔS) rescued by a URA3 plasmid bearing wild-type SUP45. We selected a subset of mutants carrying only a single mutation, or a combination of these single mutations, for this analysis. The FS1ΔS strain was then transformed with the mutant constructs and the wild-type SUP45 gene was shuffled. Our results (Figure 2) clearly demonstrated that all the mutant cells were viable at 30°C. However, all three double mutants were thermosensitive.Figure 2.

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
Related in: MedlinePlus