Release Factor eRF3 Mediates Premature Translation Termination on Polylysine-Stalled Ribosomes in Saccharomyces cerevisiae

ABSTRACT Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

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Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation

Molecular and Cellular Biology ◽

10.1128/mcb.22.10.3301-3315.2002 ◽

2002 ◽

Vol 22 (10) ◽

pp. 3301-3315 ◽

Cited By ~ 98

Author(s):

Bertrand Cosson ◽

Anne Couturier ◽

Svetlana Chabelskaya ◽

Denis Kiktev ◽

Sergey Inge-Vechtomov ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Translational Control ◽

Genetic Interaction ◽

Translation Termination ◽

Hybrid Approach ◽

Translation Efficiency ◽

Release Factor ◽

Initiation Of Translation ◽

Two Hybrid ◽

Lines Of Evidence

ABSTRACT Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 (eRF3) mutant and [PSI +] cells. This effect requires the interaction of Pab1p with eRF3. These data further strengthen the possibility that Pab1p has a role in coupling translation termination events with initiation of translation. Several lines of evidence indicate that Pab1p does not influence [PSI +] propagation. First, “[PSI +]-no-more” mutations do not affect eRF3-Pab1p two-hybrid interaction. Second, overexpression of PAB1 does not cure the [PSI +] phenotype or solubilize detectable amounts of eRF3. Third, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI +], were not modified by excess Pab1p.

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Cutting the nonsense: the degradation of PTC-containing mRNAs

Biochemical Society Transactions ◽

10.1042/bst0381615 ◽

2010 ◽

Vol 38 (6) ◽

pp. 1615-1620 ◽

Cited By ~ 77

Author(s):

Pamela Nicholson ◽

Oliver Mühlemann

Keyword(s):

Saccharomyces Cerevisiae ◽

Quality Control ◽

Control Function ◽

Translation Termination ◽

Rapid Degradation ◽

Morphological Defects ◽

Nonsense Mediated Mrna Decay ◽

Specific Factors ◽

Transcriptional Pathway ◽

Premature Translation Termination

In eukaryotes, mRNAs harbouring PTCs (premature translation-termination codons) are recognized and eliminated by NMD (nonsense-mediated mRNA decay). In addition to its quality-control function, NMD constitutes a translation-dependent post-transcriptional pathway to regulate the expression levels of physiological mRNAs. In contrast with PTC recognition, little is known about the mechanisms that trigger the rapid degradation of mammalian nonsense mRNA. Studies have shown that mammalian NMD targets can be degraded via both an SMG6 (where SMG is suppressor of morphological defects on genitalia)-dependent endonucleolytic pathway and a deadenylation and decapping-dependent exonucleolytic pathway, with the possible involvement of SMG5 and SMG7. In contrast, Drosophila melanogaster NMD is confined to the former and Saccharomyces cerevisiae NMD to the latter decay pathway. Consistent with this conclusion, mammals possess both SMG6 and SMG7, whereas D. melanogaster lacks an SMG7 homologue and yeast have no SMG6 equivalent. In the present paper, we review what is known about the degradation of PTC-containing mRNAs so far, paying particular attention to the properties of the NMD-specific factors SMG5–SMG7 and to what is known about the mechanism of degrading mRNAs after they have been committed to the NMD pathway.

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Gene Overexpression as a Tool for Identifying New trans-Acting Factors Involved in Translation Termination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/161.2.585 ◽

2002 ◽

Vol 161 (2) ◽

pp. 585-594

Author(s):

Olivier Namy ◽

Isabelle Hatin ◽

Guillaume Stahl ◽

Hongmei Liu ◽

Stephanie Barnay ◽

...

Keyword(s):

Protein Transport ◽

Stop Codon ◽

Translation Termination ◽

Spindle Pole Body ◽

Spindle Pole ◽

Release Factor ◽

Lacz Reporter Gene ◽

Pole Body ◽

Secondary Phenotypes ◽

Termination Process

Abstract In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.

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The influence of 5' codon context on translation termination in Saccharomyces cerevisiae

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1998.2570249.x ◽

1998 ◽

Vol 257 (1) ◽

pp. 249-254 ◽

Cited By ~ 44

Author(s):

Salim Mottagui-Tabar ◽

Mick F. Tuite ◽

Leif A. Isaksson

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Termination ◽

Codon Context

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Caenorhabditis elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons

Molecular and Cellular Biology ◽

10.1128/mcb.00410-07 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5630-5638 ◽

Cited By ~ 39

Author(s):

Lisa Johns ◽

Andrew Grimson ◽

Sherry L. Kuchma ◽

Carrie Loushin Newman ◽

Philip Anderson

Keyword(s):

Caenorhabditis Elegans ◽

Mammalian Cells ◽

Translation Termination ◽

Yeast Two Hybrid ◽

Eukaryotic Mrnas ◽

Nonsense Mediated Mrna Decay ◽

Endogenous Genes ◽

Alternatively Spliced ◽

Two Hybrid ◽

Premature Translation Termination

ABSTRACT Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed “nonsense-mediated mRNA decay” (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with (“marks”) those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

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Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

eLife ◽

10.7554/elife.62655 ◽

2020 ◽

Vol 9 ◽

Author(s):

Kyle Mangano ◽

Tanja Florin ◽

Xinhao Shao ◽

Dorota Klepacki ◽

Irina Chelysheva ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Stop Codon ◽

Translation Termination ◽

Bacterial Cells ◽

Release Factor ◽

Translation Arrest ◽

Genome Wide Analysis ◽

Genome Wide ◽

Exit Tunnel ◽

Unique Mechanism

Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.

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Crystal structure of a translation termination complex formed with release factor RF2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0810953105 ◽

2008 ◽

Vol 105 (50) ◽

pp. 19684-19689 ◽

Cited By ~ 159

Author(s):

A. Korostelev ◽

H. Asahara ◽

L. Lancaster ◽

M. Laurberg ◽

A. Hirschi ◽

...

Keyword(s):

Crystal Structure ◽

Translation Termination ◽

Release Factor

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Observation of Amyloid-Like Fibers of the Sup35 Protein from Saccharomyces Cerevisiae

Microscopy and Microanalysis ◽

10.1017/s1431927600035819 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 664-665

Author(s):

Anthony S. Kowal ◽

Thomas Scheibel ◽

Susan L. Lindquist

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Conformation ◽

Prion Proteins ◽

Translation Termination ◽

Polarized Light ◽

Yeast Cells ◽

Yeast Prion ◽

Epigenetic Modifier ◽

Insoluble Form

In the yeast Saccharomyces cerevisiae, [PST] acts as an epigenetic modifier of translation termination efficiency. [PSI+] can be passed through generations of yeast cells via changes in protein conformation rather than changes in DNA or RNA, and has thus been referred to as a yeast prion. The [PSI+] determinant is the Sup35 protein. Sup35 can exist in two states - soluble and insoluble. Soluble Sup35 functions in translation termination, but when insoluble, stop codons are read through, resulting in incorrect protein products.Sup35 is composed of three distinct domains, N, M, and C. The N region is rich in glutamine and asparagine and is required for the [PST] phenotype to exist. M is a highly charged domain, and no specific function has been assigned to it. C is essential in yeast, as it is responsible for translation termination. The insoluble form of Sup35 has characteristics reminiscent of other prion proteins - in vitro it binds to the dye Congo Red and it exhibits apple green birefringence in polarized light.

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