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

eLife ◽  
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.

scholarly journals Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

2020 ◽  
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

AbstractBiochemical 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 revealed that 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 expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate development of new medicines and research tools for genome exploration.

Influence of nascent polypeptide positive charges on translation dynamics

2020 ◽  
Vol 477 (15) ◽  
pp. 2921-2934
Author(s):  
Rodrigo D. Requião ◽  
Géssica C. Barros ◽  
Tatiana Domitrovic ◽  
Fernando L. Palhano
Keyword(s):  
Mrna Decay ◽  
Translation Termination ◽  
Published Data ◽  
Translation Efficiency ◽  
Amino Acid Residues ◽  
High Concentration ◽  
Strong Argument ◽  
Translation Arrest ◽  
Exit Tunnel ◽  
Positively Charged

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

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

Genetics ◽  
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.

scholarly journals Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (8) ◽  
pp. 1378-1387 ◽  
Author(s):  
Adam K. Kallmeyer ◽  
Kim M. Keeling ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Number Of Factors ◽  
Stop Codon Recognition

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.

scholarly journals PABP enhances release factor recruitment and stop codon recognition during translation termination

2016 ◽  
Vol 44 (16) ◽  
pp. 7766-7776 ◽  
Author(s):  
Alexandr Ivanov ◽  
Tatyana Mikhailova ◽  
Boris Eliseev ◽  
Lahari Yeramala ◽  
Elizaveta Sokolova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Stop Codon Recognition

Identification of amino acids responsible for stop codon recognition for polypeptide chain release factor

2013 ◽  
Vol 91 (3) ◽  
pp. 155-164 ◽  
Author(s):  
Lijun Xu ◽  
Yanrong Hao ◽  
Cui Li ◽  
Quan Shen ◽  
Baofeng Chai ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Reassignment ◽  
Eukaryotic Translation ◽  
Codon Recognition ◽  
Terminal Domain ◽  
Class 1 ◽  
Stop Codon Recognition ◽  
Stop Codon Reassignment

One factor involved in eukaryotic translation termination is class 1 release factor in eukaryotes (eRF1), which functions to decode stop codons. Variant code species, such as ciliates, frequently exhibit altered stop codon recognition. Studies revealed that some class-specific residues in the eRF1 N-terminal domain are responsible for stop codon reassignment in ciliates. Here, we investigated the effects on stop codon recognition of chimeric eRF1s containing the N-terminal domain of Euplotes octocarinatus and Blepharisma japonicum eRF1 fused to Saccharomyces cerevisiae M and C domains using dual luciferase read-through assays. Mutation of class-specific residues in different eRF1 classes was also studied to identify key residues and motifs involved in stop codon decoding. As expected, our results demonstrate that 3 pockets within the eRF1 N-terminal domain were involved in decoding stop codon nucleotides. However, allocation of residues to each pocket was revalued. Our data suggest that hydrophobic and class-specific surface residues participate in different functions: modulation of pocket conformation and interaction with stop codon nucleotides, respectively. Residues conserved across all eRF1s determine the relative orientation of the 3 pockets according to stop codon nucleotides. However, quantitative analysis of variant ciliate and yeast eRF1 point mutants did not reveal any correlation between evolutionary conservation of class-specific residues and termination-related functional specificity and was limited in elucidating a detailed mechanism for ciliate stop codon reassignment. Thus, based on isolation of suppressor tRNAs from Euplotes and Tetrahymena, we propose that stop codon reassignment in ciliates may be controlled by cooperation between eRF1 and suppressor tRNAs.

scholarly journals Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

2020 ◽  
Author(s):  
Alexey Shuvalov ◽  
Ekaterina Shuvalova ◽  
Nikita Biziaev ◽  
Elizaveta Sokolova ◽  
Konstantin Evmenov ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Release Factor ◽  
Viral Mrnas ◽  
Codon Recognition ◽  
Free Translation ◽  
Stop Codon Recognition ◽  
A Cell

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

scholarly journals Mutations in Eukaryotic Release Factors 1 and 3 Act as General Nonsense Suppressors in Drosophila

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 601-612
Author(s):  
Anna T Chao ◽  
Herman A Dierick ◽  
Tracie M Addy ◽  
Amy Bejsovec
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Nonsense Suppression ◽  
Release Factor ◽  
Nonsense Mutations ◽  
Stop Codons ◽  
Larval Stages ◽  
Mutant Phenotypes ◽  
Polypeptide Chains ◽  
Nonsense Suppressors

Abstract In a screen for suppressors of the Drosophila winglessPE4 nonsense allele, we isolated mutations in the two components that form eukaryotic release factor. eRF1 and eRF3 comprise the translation termination complex that recognizes stop codons and catalyzes the release of nascent polypeptide chains from ribosomes. Mutations disrupting the Drosophila eRF1 and eRF3 show a strong maternal-effect nonsense suppression due to readthrough of stop codons and are zygotically lethal during larval stages. We tested nonsense mutations in wg and in other embryonically acting genes and found that different stop codons can be suppressed but only a subset of nonsense alleles are subject to suppression. We suspect that the context of the stop codon is significant: nonsense alleles sensitive to suppression by eRF1 and eRF3 encode stop codons that are immediately followed by a cytidine. Such suppressible alleles appear to be intrinsically weak, with a low level of readthrough that is enhanced when translation termination is disrupted. Thus the eRF1 and eRF3 mutations provide a tool for identifying nonsense alleles that are leaky. Our findings have important implications for assigning null mutant phenotypes and for selecting appropriate alleles to use in suppressor screens.

scholarly journals A Genome-Wide Analysis of RNA Pseudoknots That Stimulate Efficient −1 Ribosomal Frameshifting or Readthrough in Animal Viruses

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Xiaolan Huang ◽  
Qiang Cheng ◽  
Zhihua Du
Keyword(s):  
Large Scale ◽  
Stop Codon ◽  
Ribosomal Frameshifting ◽  
Viral Genomes ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Rna Pseudoknots ◽  
Rna Pseudoknot ◽  
Stop Codon Readthrough

Programmed −1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at thegag-polframeshift junction of HIV1 harbors potential elaborated pseudoknots encompassing the frameshift site. A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event.

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