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 eR… Show more
“…The type and position of amino acid substitution can also affect the phenotype. The best studied model systems, such as E. coli and S. cerevisiae , have revealed complex determinants extending at least six base pairs downstream of the stop codon itself [ e.g., (Poole et al 1998; Namy et al 2001; Poole et al 2003; Hatin et al 2009)]. Our findings are consistent with such mechanisms operating in S. pombe , too.…”
In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI+] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.
“…The type and position of amino acid substitution can also affect the phenotype. The best studied model systems, such as E. coli and S. cerevisiae , have revealed complex determinants extending at least six base pairs downstream of the stop codon itself [ e.g., (Poole et al 1998; Namy et al 2001; Poole et al 2003; Hatin et al 2009)]. Our findings are consistent with such mechanisms operating in S. pombe , too.…”
In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI+] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.
“…This study extended the cavity model by suggesting that conformational changes hinging around the TASNIKS motif optimize the relative orientation of the cavities for decoding of UAA/UAG on one hand and UGA on the other. Lastly, based on the results of an anti-suppressor screen, Hatin et al (6) identified two new regions important for stop codon decoding they termed pocket one and two (Figure 7), which were proposed to be involved in maintaining an overall structural frame-work of the N-domain that is optimal for eRF1 function in translation termination. Figure 7.Stop codon specificity of read-through in eRF1 N-domain mutants.…”
Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
“…The shape of human eRF1 resembles a tRNA, with functional motifs targeting both the peptidyl transferase center and the decoding site. eRF1 recognizes all three stop codons through an extended area in its N-terminal domain [21][22][23], although it has recently become clear that eRF1 acts differently depending on the type of codon; its behavior with UGA being different from that with UAA or UAG [21,24]. When a stop codon is recognized, the highly conserved Gly-Gly-Gln (GGQ) motif in the central domain of eRF1 triggers peptidyl-tRNA hydrolysis by activating the peptidyl transferase center of the ribosome [25][26][27].…”
Section: Mechanisms Of Translation Terminationmentioning
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