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Eukaryotic release factor 3 is required for multiple turnovers of peptide release catalysis by eukaryotic release factor 1.

Eyler DE, Wehner KA, Green R - J. Biol. Chem. (2013)

Bottom Line: This effect was generalizable across all stop codons and in a variety of contexts.These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release.A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

View Article: PubMed Central - PubMed

Affiliation: From the Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

ABSTRACT
Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

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Stop codon and the distal nucleotide at position +4 have small effects on the rate of peptide release.A, the rate constant for peptide release at saturating release factor concentrations depends slightly on the stop codon and the nucleotide at position +4. The white bars indicate the rate of peptide release mediated by eRF1:eRF3:GTP, whereas the black bars indicate the rate of peptide release mediated by eRF1 alone. B, the observed rates of multiple turnover peptide release by eRF1 varies <2-fold across a subset of stop codons and +4 nucleotides. Reactions were carried out as in Fig. 1B. Observed rates are plotted; error bars represent the S.E. C, the observed rates of multiple turnover peptide release by eRF1 and eRF3 depend slightly on stop codon and the nucleotide at position +4. Reactions were carried out as described in Fig. 1B. Observed rates are plotted; error bars represent the range.
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Figure 2: Stop codon and the distal nucleotide at position +4 have small effects on the rate of peptide release.A, the rate constant for peptide release at saturating release factor concentrations depends slightly on the stop codon and the nucleotide at position +4. The white bars indicate the rate of peptide release mediated by eRF1:eRF3:GTP, whereas the black bars indicate the rate of peptide release mediated by eRF1 alone. B, the observed rates of multiple turnover peptide release by eRF1 varies <2-fold across a subset of stop codons and +4 nucleotides. Reactions were carried out as in Fig. 1B. Observed rates are plotted; error bars represent the S.E. C, the observed rates of multiple turnover peptide release by eRF1 and eRF3 depend slightly on stop codon and the nucleotide at position +4. Reactions were carried out as described in Fig. 1B. Observed rates are plotted; error bars represent the range.

Mentions: Previous studies had suggested that the sequence context of the stop codon differentially impacts recognition (i.e. binding) or catalysis by eRF1 and eRF1:eRF3 (23). Because our peptide release experiments had utilized only one stop codon in one sequence context, we asked whether the modest stimulation of peptide release by eRF3 could be generalized to other stop codons and contexts. For this analysis, we generated termination complexes containing each of the three stop codons (UAA, UAG, and UGA) in the A site followed by the four different nucleotides at position +4 (a total of 12 sequences). Rate constants for peptide release were then determined using both saturating eRF1 alone, as well as saturating eRF1 in combination with saturating levels of eRF3 and GTP (Fig. 2A). Rates of eRF1-only peptide release ranged from 0.006 s−1 on UAG C to 0.014 s−1 on UAA A, a range of 2.3-fold. Addition of eRF3 yielded increased overall rates ranging between 0.03 s−1 on UAG A and 0.06 s−1 on UAA C, a range of 2-fold. The stimulation afforded by eRF3 was between 3- and 10-fold on UGA A and UGA C, respectively, whereas the average stimulation by eRF3 was 5-fold. These modest effects of eRF3 on codon recognition in the in vitro system do not correlate particularly well with the earlier in vivo studies (23).


Eukaryotic release factor 3 is required for multiple turnovers of peptide release catalysis by eukaryotic release factor 1.

Eyler DE, Wehner KA, Green R - J. Biol. Chem. (2013)

Stop codon and the distal nucleotide at position +4 have small effects on the rate of peptide release.A, the rate constant for peptide release at saturating release factor concentrations depends slightly on the stop codon and the nucleotide at position +4. The white bars indicate the rate of peptide release mediated by eRF1:eRF3:GTP, whereas the black bars indicate the rate of peptide release mediated by eRF1 alone. B, the observed rates of multiple turnover peptide release by eRF1 varies <2-fold across a subset of stop codons and +4 nucleotides. Reactions were carried out as in Fig. 1B. Observed rates are plotted; error bars represent the S.E. C, the observed rates of multiple turnover peptide release by eRF1 and eRF3 depend slightly on stop codon and the nucleotide at position +4. Reactions were carried out as described in Fig. 1B. Observed rates are plotted; error bars represent the range.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795251&req=5

Figure 2: Stop codon and the distal nucleotide at position +4 have small effects on the rate of peptide release.A, the rate constant for peptide release at saturating release factor concentrations depends slightly on the stop codon and the nucleotide at position +4. The white bars indicate the rate of peptide release mediated by eRF1:eRF3:GTP, whereas the black bars indicate the rate of peptide release mediated by eRF1 alone. B, the observed rates of multiple turnover peptide release by eRF1 varies <2-fold across a subset of stop codons and +4 nucleotides. Reactions were carried out as in Fig. 1B. Observed rates are plotted; error bars represent the S.E. C, the observed rates of multiple turnover peptide release by eRF1 and eRF3 depend slightly on stop codon and the nucleotide at position +4. Reactions were carried out as described in Fig. 1B. Observed rates are plotted; error bars represent the range.
Mentions: Previous studies had suggested that the sequence context of the stop codon differentially impacts recognition (i.e. binding) or catalysis by eRF1 and eRF1:eRF3 (23). Because our peptide release experiments had utilized only one stop codon in one sequence context, we asked whether the modest stimulation of peptide release by eRF3 could be generalized to other stop codons and contexts. For this analysis, we generated termination complexes containing each of the three stop codons (UAA, UAG, and UGA) in the A site followed by the four different nucleotides at position +4 (a total of 12 sequences). Rate constants for peptide release were then determined using both saturating eRF1 alone, as well as saturating eRF1 in combination with saturating levels of eRF3 and GTP (Fig. 2A). Rates of eRF1-only peptide release ranged from 0.006 s−1 on UAG C to 0.014 s−1 on UAA A, a range of 2.3-fold. Addition of eRF3 yielded increased overall rates ranging between 0.03 s−1 on UAG A and 0.06 s−1 on UAA C, a range of 2-fold. The stimulation afforded by eRF3 was between 3- and 10-fold on UGA A and UGA C, respectively, whereas the average stimulation by eRF3 was 5-fold. These modest effects of eRF3 on codon recognition in the in vitro system do not correlate particularly well with the earlier in vivo studies (23).

Bottom Line: This effect was generalizable across all stop codons and in a variety of contexts.These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release.A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

View Article: PubMed Central - PubMed

Affiliation: From the Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

ABSTRACT
Eukaryotic peptide release factor 3 (eRF3) is a conserved, essential gene in eukaryotes implicated in translation termination. We have systematically measured the contribution of eRF3 to the rates of peptide release with both saturating and limiting levels of eukaryotic release factor 1 (eRF1). Although eRF3 modestly stimulates the absolute rate of peptide release (∼5-fold), it strongly increases the rate of peptide release when eRF1 is limiting (>20-fold). This effect was generalizable across all stop codons and in a variety of contexts. Further investigation revealed that eRF1 remains associated with ribosomal complexes after peptide release and subunit dissociation and that eRF3 promotes the dissociation of eRF1 from these post-termination complexes. These data are consistent with models where eRF3 principally affects binding interactions between eRF1 and the ribosome, either prior to or subsequent to peptide release. A role for eRF3 as an escort for eRF1 into its fully accommodated state is easily reconciled with its close sequence similarity to the translational GTPase EFTu.

Show MeSH
Related in: MedlinePlus