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. 2004 Sep;24(17):7769-78.
doi: 10.1128/MCB.24.17.7769-7778.2004.

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination

Joe Salas-Marco  1 , David M Bedwell
Affiliations

GTP hydrolysis by eRF3 facilitates stop codon decoding during eukaryotic translation termination

Joe Salas-Marco et al. Mol Cell Biol. 2004 Sep.

Abstract

Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

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Figures

FIG. 1.
FIG. 1.
Diagram showing the location of eRF3 mutations analyzed in this study. (A) Schematic map of eRF3 showing the mutations introduced into the C-terminal GTPase domain. (B) Steady-state levels of wild-type (WT) and mutant forms of eRF3 as determined by Western blot analysis. Levels of eRF3 were measured in a sup35Δ strain (YBD498) expressing the indicated wild-type or mutant forms of eRF3 from a low-copy-number plasmid. Due to the inability of eRF3-H348L to support cell viability, it was coexpressed with a derivative of wild-type eRF3 (eRF3*) containing a small internal deletion (amino acids residues 21 to 67) in the N region. Twenty-five micrograms of total protein was loaded per lane.
FIG. 2.
FIG. 2.
Dual luciferase readthrough reporter constructs.
FIG. 3.
FIG. 3.
eRF3 GTPase assays. GTPase assays were carried out with various combinations of purified eRF3-ΔNM, eRF1, and salt-washed ribosomes. The graph indicates the GTP hydrolysis associated with 3 pmol each of eRF3-ΔNM and eRF1 (triangles), eRF3-ΔNM and ribosomes (squares), eRF1 and ribosomes (diamonds), and eRF3-ΔNM, eRF1, and ribosomes (circles).
FIG. 4.
FIG. 4.
Kinetic analysis of the GTPase activity of wild-type (WT) and mutant forms of eRF3. Reactions contained 3 pmol each of eRF1, eRF3-ΔNM, and ribosomes.
FIG. 5.
FIG. 5.
Reduced steady-state level of eRF1 following its expression from the CUP1 promoter as determined by Western blot analysis. Reduced expression of eRF1 was driven from the CUP1 promoter (PCUP1) in a sup45Δ strain, while the control strain expressed eRF1 from its natural promoter (PSUP45) at its genomic locus. Both strains were grown in SMD medium containing 50 μM EDTA to specifically reduce eRF1 expression from the CUP1 promoter. Twenty-five micrograms of total protein was loaded per lane.
FIG. 6.
FIG. 6.
Reduced steady-state level of eRF3 does not alter the amount of eRF1 associated with ribosomes. Western blots indicating total and ribosome-associated eRF3 levels (A) and total and ribosome-associated eRF1 levels (B) are shown. Twenty-five micrograms of total protein was loaded per lane. Reduced expression of eRF3 was driven from the CUP1 promoter (PCUP1) in a sup35Δ strain, while the control strain expressed eRF3 from its natural promoter (PSUP35) at its genomic locus. Both strains were grown in SMD medium containing 50 μM EDTA to specifically reduce eRF3 expression from the CUP1 promoter.
FIG. 7.
FIG. 7.
Introduction of a multicopy plasmid expressing the SUP45 gene from its own promoter leads to an increased steady-state level of eRF1. WT, wild type.
FIG. 8.
FIG. 8.
Model illustrating how GTP hydrolysis by eRF3 may enhance the decoding of termination signals during translation termination.
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References

    1. Alberts, B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002. Molecular Biology of the Cell, 4th ed., p. 616. Garland Science, New York, N.Y.
    1. Anborgh, P. H., R. H. Cool, F. Gumusel, K. Harmark, E. Jacquet, A. Weijland, M. Y. Mistou, and A. Parmeggiani. 1991. Structure-function relationships of elongation factor Tu as studied by mutagenesis. Biochimie 73:1051-1059. - PubMed
    1. Bertram, G., H. A. Bell, D. W. Ritchie, G. Fullerton, and I. Stansfield. 2000. Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA 6:1236-1247. - PMC - PubMed
    1. Bonetti, B., L. Fu, J. Moon, and D. M. Bedwell. 1995. The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J. Mol. Biol. 251:334-345. - PubMed
    1. Brown, C. M., P. A. Stockwell, C. N. Trotman, and W. P. Tate. 1990. Sequence analysis suggests that tetra-nucleotides signal the termination of protein synthesis in eukaryotes. Nucleic Acids Res. 18:6339-6345. - PMC - PubMed

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