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. 2013 Oct 11;288(41):29530-8.
doi: 10.1074/jbc.M113.487090. Epub 2013 Aug 20.

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

Daniel E Eyler  1 , Karen A Wehner, Rachel Green
Affiliations

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

Daniel E Eyler et al. J Biol Chem. .

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.

Keywords: Protein Synthesis; Ribosomes; Translation; Translation Control; Translation Release Factors; Translational Control.

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Figures

FIGURE 1.
FIGURE 1.
eRF3 markedly stimulates the rate of multiple turnover peptide release by eRF1. A, the rate constant for peptide release at saturating release factor concentrations depends on which factors and nucleotides are added. 1:GTP indicates eRF1 (1 μm) and GTP, 1:3:GTP indicates eRF1, eRF3 (2 μm), and 1 mm GTP, etc. B, multiple turnover peptide release depends on eRF3 and GTP. Limiting (2 nm) eRF1 was incubated with excess pre-termination complex (∼70 nm) and the fraction of dipeptide released was monitored as a function of time. eRF3 was added at saturating levels when indicated; nucleotides were at 1 mm. C, the rate of single turnover subunit dissociation does not depend on eRF3. Termination complexes were prepared with a 32P-labeled tRNA in the P site, and the fraction of subunits dissociated over time was monitored by native gel analysis. Factors were added at saturating concentrations as indicated in the legend. D, multiple turnover subunit dissociation depends on eRF3 and GTP. Termination complexes were prepared as in C and reacted with limiting eRF1, saturating eRF3, and nucleotides as in B.
FIGURE 2.
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.
FIGURE 3.
FIGURE 3.
eRF1 remains associated with ribosomal subunits after peptide release and subunit dissociation. A, eRF1 is detected throughout the gradient in the absence of eRF3. Termination complexes (∼70 nm) were reacted with stoichiometric eRF1, with or without eRF3 (1 μm), and separated by sucrose density gradient centrifugation. A trace of absorbance at 254 nm is shown, and the positions of the ribosomal components are labeled. Fractions were collected as indicated by the tick marks in the absorbance trace and analyzed for the presence of eRF1 by Western blotting with standard ECL techniques with an antibody against eRF1. eRF1 is almost exclusively at the top of the gradient in the presence of eRF3 but co-sediments substantially with the 40S, 60S, and 80S peaks in the absence of eRF3. B, eRF1 is detected in the 40S and 60S peaks in the absence of eRF3. Termination complexes were prepared and reacted with eRF1 and/or eRF3, and sucrose density gradient centrifugation was performed as described in A. The results of a quantitative Western blot are shown below the 254 nm absorbance trace. “No ribos“ indicates eRF1 was centrifuged without termination complexes as a negative control. Western blotting was performed using fluorescently labeled secondary antibodies (LICOR). The signal was quantitated using Odyssey and plotted on the y axis in arbitrary units of fluorescence intensity. Note that the y axis is broken to show the fluorescence in fractions 1 and 2.
FIGURE 4.
FIGURE 4.
Depletion of eRF3 in vivo results in redistribution of eRF1 on polysome gradients. A, Western blot analysis of total cellular lysates prepared from wild type ERF3 (the SUP35 gene) and pGAL1::erf3ΔN253 strains grown for 8 h in galactose (permissive for both) or glucose (erf3ΔN253 depletion). Equal A260 units of each lysate were loaded on the gel. The asterisk indicates a background band. B, cellular lysates prepared from wild type ERF3 (black line) and pGAL1::erf3ΔN253 (red line) strains grown for 8 h in glucose (erf3pΔN253 depletion). Equal A260 units of each lysate were separated on sucrose density gradients as described under “Experimental Procedures.” Absorbance peaks (254 nm) that correspond to the ribosomal 40S and 60S subunits as well as 80S monosomes and polysomes are indicated. Fraction 1 is the top of the gradient, and fraction 15 is the bottom of the gradient. C, Western blot analysis of the sedimentation of eRF1 and Rps6p during sucrose gradient analysis of wild type ERF3 and pGAL1::erf3ΔN253 strains grown in glucose. The asterisk indicates a background band observed in fractions 1 and 2 prior to detection with the rpS6 antibody. D, quantitation of the percent of total protein found in each sucrose gradient fraction for the Western blots shown in B.
FIGURE 5.
FIGURE 5.
Model of the roles of eRF1 and eRF3 in eukaryotic termination. A simplified model for eukaryotic termination, emphasizing the steps at which eRF1 may be influenced by eRF3. krelease and ksplitting refer to the processes of peptide release and subunit dissociation, respectively. These two steps could be directly measured in our kinetic analysis; binding steps (indicated with equilibria) have not been directly monitored here.
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