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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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Structural model of mutations identified in eRF1. (A) The left panel shows P1 in the same orientation as shown in Supplementary Figure S3B. Wild-type amino acids are displayed in yellow, whereas mutated residues are shown in red and orange. The changes induced by these mutations at the surface of the protein are shown in transparency surface mode. The right panel is rotated as indicated to give a more detailed view. (B) The left panel is a surface representation of P2 with labeling of the four residues creating the pocket 2. The three residues found mutated are shown in yellow; the residue with no associated mutation is shown in blue. The right panel is a magnification of the image showing P2 with the same orientation. The residue Arg76 found in our screen is shown in red.
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Figure 4: Structural model of mutations identified in eRF1. (A) The left panel shows P1 in the same orientation as shown in Supplementary Figure S3B. Wild-type amino acids are displayed in yellow, whereas mutated residues are shown in red and orange. The changes induced by these mutations at the surface of the protein are shown in transparency surface mode. The right panel is rotated as indicated to give a more detailed view. (B) The left panel is a surface representation of P2 with labeling of the four residues creating the pocket 2. The three residues found mutated are shown in yellow; the residue with no associated mutation is shown in blue. The right panel is a magnification of the image showing P2 with the same orientation. The residue Arg76 found in our screen is shown in red.

Mentions: Both release factors eRF1 and eRF3 are key components of the termination process. The eRF1 protein can be divided into three functional domains. Domain 1 binds the stop codon directly in the ribosomal A-site. Domain 2 interacts with the peptidyl transferase center leading to the hydrolysis of the last peptidyl-tRNA bond through the GGQ motif (10,26). Little structural data is available for domain 3, however, in S. pombe, the last 11 residues are necessary for the binding of eRF3 (6,27–29). We developed an original approach to identify new mutants displaying an anti-suppressor phenotype in a wild-type genetic background by taking advantage of the weak termination efficiency of stop codons present in readthrough sequences. Using this screen, we identified 131 mutations distributed throughout the SUP45 gene. All single mutants were viable at either 30°C or 37°C in a strain carrying deletion of SUP45 and did not display any obvious phenotype. Thus, the increase in termination efficiency had no deleterious effect on yeast viability. However, these mutants were not viable in the absence of eRF3, demonstrating that these mutations do not compensate for loss of eRF3 by directly activating eRF1 (data not shown). We demonstrated most of the mutants had an equal amount of eRF1 protein and showed a substantial increase in termination efficiency, demonstrating a direct effect on termination mechanisms. Moreover, we showed that the combination of single mutations, identified separately in domains 1 and 3, affects termination efficiency differently, depending on the mutations and stop codon involved. These findings suggest that mutations in the various domains modify termination efficiency through their effects on different mechanisms. We were able to localize these mutations on the 3D structure of the human eRF1 using Pymol software (see Materials and methods section). All mutations found in domains 1 and 2 appear to modify the surface of the protein, whereas the mutations found in domain 3 have negligible effects on the surface of the protein (Figure S4). Mutations located in domain 1 are subdivided into three distinct groups (Figure 4). The first group (affecting Asn27, Asp96 and Glu104) constitutes a pocket 1 (P1) at the top of this domain (Figure 4A), lying very close to the regions directly involved in the stop codon recognition. A ‘pocket model’ has been proposed for stop codon recognition by eRF1 (4,11). An important feature of this model is the H-bond formed between residues Glu55 and Tyr122, which would maintain the two remote regions of domain 1 (the two NIKS domain-containing helices, and the beta-strand on the opposite side, represented in dark blue in Figure S3) in close proximity to each other. The new region P1 is continuous with this functional area. Both these regions could together accommodate the mRNA for proper orientation of eRF1 in the A-site. As observed in Figure 4, the introduction of either His or Tyr at position 27 should exert a similar structural bulk effect on the protein surface. These mutations would deepen pocket 1 and probably strengthen interactions with the helix domain.Figure 4.


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)

Structural model of mutations identified in eRF1. (A) The left panel shows P1 in the same orientation as shown in Supplementary Figure S3B. Wild-type amino acids are displayed in yellow, whereas mutated residues are shown in red and orange. The changes induced by these mutations at the surface of the protein are shown in transparency surface mode. The right panel is rotated as indicated to give a more detailed view. (B) The left panel is a surface representation of P2 with labeling of the four residues creating the pocket 2. The three residues found mutated are shown in yellow; the residue with no associated mutation is shown in blue. The right panel is a magnification of the image showing P2 with the same orientation. The residue Arg76 found in our screen is shown in red.
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Figure 4: Structural model of mutations identified in eRF1. (A) The left panel shows P1 in the same orientation as shown in Supplementary Figure S3B. Wild-type amino acids are displayed in yellow, whereas mutated residues are shown in red and orange. The changes induced by these mutations at the surface of the protein are shown in transparency surface mode. The right panel is rotated as indicated to give a more detailed view. (B) The left panel is a surface representation of P2 with labeling of the four residues creating the pocket 2. The three residues found mutated are shown in yellow; the residue with no associated mutation is shown in blue. The right panel is a magnification of the image showing P2 with the same orientation. The residue Arg76 found in our screen is shown in red.
Mentions: Both release factors eRF1 and eRF3 are key components of the termination process. The eRF1 protein can be divided into three functional domains. Domain 1 binds the stop codon directly in the ribosomal A-site. Domain 2 interacts with the peptidyl transferase center leading to the hydrolysis of the last peptidyl-tRNA bond through the GGQ motif (10,26). Little structural data is available for domain 3, however, in S. pombe, the last 11 residues are necessary for the binding of eRF3 (6,27–29). We developed an original approach to identify new mutants displaying an anti-suppressor phenotype in a wild-type genetic background by taking advantage of the weak termination efficiency of stop codons present in readthrough sequences. Using this screen, we identified 131 mutations distributed throughout the SUP45 gene. All single mutants were viable at either 30°C or 37°C in a strain carrying deletion of SUP45 and did not display any obvious phenotype. Thus, the increase in termination efficiency had no deleterious effect on yeast viability. However, these mutants were not viable in the absence of eRF3, demonstrating that these mutations do not compensate for loss of eRF3 by directly activating eRF1 (data not shown). We demonstrated most of the mutants had an equal amount of eRF1 protein and showed a substantial increase in termination efficiency, demonstrating a direct effect on termination mechanisms. Moreover, we showed that the combination of single mutations, identified separately in domains 1 and 3, affects termination efficiency differently, depending on the mutations and stop codon involved. These findings suggest that mutations in the various domains modify termination efficiency through their effects on different mechanisms. We were able to localize these mutations on the 3D structure of the human eRF1 using Pymol software (see Materials and methods section). All mutations found in domains 1 and 2 appear to modify the surface of the protein, whereas the mutations found in domain 3 have negligible effects on the surface of the protein (Figure S4). Mutations located in domain 1 are subdivided into three distinct groups (Figure 4). The first group (affecting Asn27, Asp96 and Glu104) constitutes a pocket 1 (P1) at the top of this domain (Figure 4A), lying very close to the regions directly involved in the stop codon recognition. A ‘pocket model’ has been proposed for stop codon recognition by eRF1 (4,11). An important feature of this model is the H-bond formed between residues Glu55 and Tyr122, which would maintain the two remote regions of domain 1 (the two NIKS domain-containing helices, and the beta-strand on the opposite side, represented in dark blue in Figure S3) in close proximity to each other. The new region P1 is continuous with this functional area. Both these regions could together accommodate the mRNA for proper orientation of eRF1 in the A-site. As observed in Figure 4, the introduction of either His or Tyr at position 27 should exert a similar structural bulk effect on the protein surface. These mutations would deepen pocket 1 and probably strengthen interactions with the helix domain.Figure 4.

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