Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• Klíčová slova
• DENR, MCTS1, Tma, eIF2D, reinitiation, ribosome recycling,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• MeSH
• iniciace translace peptidového řetězce MeSH
• malé podjednotky ribozomu eukaryotické * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• otevřené čtecí rámce * MeSH
• proteosyntéza MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• transkripční faktory bZIP * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transkripční faktory bZIP * MeSH

All proteins in living organisms are produced in ribosomes that facilitate the translation of genetic information into a sequence of amino acid residues. During translation, the ribosome undergoes initiation, elongation, termination, and recycling. In fact, peptide bonds are formed only during the elongation phase, which comprises periodic association of transfer RNAs and multiple auxiliary proteins with the ribosome and the addition of an amino acid to the nascent polypeptide one at a time. The protein spends a considerable amount of time attached to the ribosome. Here, we conceptually divide this portion of the protein lifetime into three stages. We define each stage on the basis of the position of the N-terminus of the nascent polypeptide within the ribosome exit tunnel and the context of the catalytic center. We argue that nascent polypeptides experience a variety of forces that determine how they translocate through the tunnel and interact with the tunnel walls. We review current knowledge about nascent polypeptide translocation and identify several white spots in our understanding of the birth of proteins.

• Klíčová slova
• antibiotics, cotranslational events, protein folding, ribosome, translation,
• MeSH
• lidé MeSH
• proteosyntéza * MeSH
• ribozomy * metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these 2 isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80S initiation complex.

• Klíčová slova
• ABCE1, cryo-EM, recycling, ribosome, translation,
• MeSH
• ABC transportéry chemie   metabolismus   MeSH
• elongační faktory MeSH
• hydrolýza MeSH
• iniciace translace peptidového řetězce * MeSH
• iniciační faktory metabolismus   MeSH
• králíci MeSH
• molekulární modely MeSH
• nukleosidy chemie   MeSH
• ribozomy metabolismus   MeSH
• terminace translace peptidového řetězce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ABC transportéry MeSH
• elongační faktory MeSH
• iniciační faktory MeSH
• nukleosidy MeSH

During translation termination in bacteria, the release factors RF1 and RF2 are recycled from the ribosome by RF3. While high-resolution structures of the individual termination factors on the ribosome exist, direct structural insight into how RF3 mediates dissociation of the decoding RFs has been lacking. Here we have used the Apidaecin 137 peptide to trap RF1 together with RF3 on the ribosome and visualize an ensemble of termination intermediates using cryo-electron microscopy. Binding of RF3 to the ribosome induces small subunit (SSU) rotation and swivelling of the head, yielding intermediate states with shifted P-site tRNAs and RF1 conformations. RF3 does not directly eject RF1 from the ribosome, but rather induces full rotation of the SSU that indirectly dislodges RF1 from its binding site. SSU rotation is coupled to the accommodation of the GTPase domain of RF3 on the large subunit (LSU), thereby promoting GTP hydrolysis and dissociation of RF3 from the ribosome.

• MeSH
• elektronová kryomikroskopie MeSH
• Escherichia coli genetika   metabolismus   MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• kationické antimikrobiální peptidy farmakologie   MeSH
• konformace proteinů MeSH
• peptidy - faktory ukončení metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• proteosyntéza účinky léků   MeSH
• ribozomální proteiny metabolismus   MeSH
• ribozomy metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• simulace molekulového dockingu MeSH
• terminace translace peptidového řetězce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• velké podjednotky ribozomu bakteriální metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• apidaecin MeSH Prohlížeč
• GTP-fosfohydrolasy MeSH
• kationické antimikrobiální peptidy MeSH
• peptidy - faktory ukončení MeSH
• prfA protein, E coli MeSH Prohlížeč
• prfC protein, E coli MeSH Prohlížeč
• proteiny z Escherichia coli MeSH
• ribozomální proteiny MeSH
• RNA transferová MeSH

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

• MeSH
• ABC transportéry chemie   genetika   metabolismus   MeSH
• arginin metabolismus   MeSH
• delece genu MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• genetické vektory chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• kodon chemie   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lysin metabolismus   MeSH
• molekulární modely MeSH
• priony chemie   genetika   metabolismus   MeSH
• regulace genové exprese u hub * MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• terminace translace peptidového řetězce * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportéry MeSH
• arginin MeSH
• kodon MeSH
• lysin MeSH
• New1 protein, S cerevisiae MeSH Prohlížeč
• priony MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.

• MeSH
• biokatalýza MeSH
• biologické modely MeSH
• dimerizace MeSH
• konformace proteinů MeSH
• messenger RNA genetika   metabolismus   ultrastruktura   MeSH
• podjednotky ribozomu metabolismus   MeSH
• proteiny z Escherichia coli chemie   metabolismus   ultrastruktura   MeSH
• ribozomy metabolismus   ultrastruktura   MeSH
• stabilita RNA MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• messenger RNA MeSH
• proteiny z Escherichia coli MeSH

Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA-protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.

• MeSH
• 5' nepřekládaná oblast MeSH
• elongace translace peptidového řetězce MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genetické techniky MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• otevřené čtecí rámce * MeSH
• regulace genové exprese * MeSH
• ribozomy metabolismus   MeSH
• terminace translace peptidového řetězce MeSH
• terminační kodon MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 3 MeSH
• terminační kodon MeSH

Nucleic acid sequence complementarity underlies many fundamental biological processes. Although first noticed a long time ago, sequence complementarity between mRNAs and ribosomal RNAs still lacks a meaningful biological interpretation. Here we used statistical analysis of large-scale sequence data sets and high-throughput computing to explore complementarity between 18S and 28S rRNAs and mRNA 3' UTR sequences. By the analysis of 27,646 full-length 3' UTR sequences from 14 species covering both protozoans and metazoans, we show that the computed 18S rRNA complementarity creates an evolutionarily conserved localization pattern centered around the ribosomal mRNA entry channel, suggesting its biological relevance and functionality. Based on this specific pattern and earlier data showing that post-termination 80S ribosomes are not stably anchored at the stop codon and can migrate in both directions to codons that are cognate to the P-site deacylated tRNA, we propose that the 18S rRNA-mRNA complementarity selectively stabilizes post-termination ribosomal complexes to facilitate ribosome recycling. We thus demonstrate that the complementarity between 18S rRNA and 3' UTRs has a non-random nature and very likely carries information with a regulatory potential for translational control.

• Klíčová slova
• large-scale data set, metazoan 18S rRNA–mRNA 3′ UTRs complementarity, ribosomal recycling, statistics, translation termination,
• MeSH
• 3' nepřekládaná oblast * MeSH
• kodon MeSH
• proteosyntéza fyziologie   MeSH
• RNA ribozomální chemie   fyziologie   MeSH
• terminátorové oblasti (genetika) * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3' nepřekládaná oblast * MeSH
• kodon MeSH
• RNA ribozomální MeSH

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

• MeSH
• antibakteriální látky farmakologie   metabolismus   MeSH
• antibiotická rezistence MeSH
• linkosamidy farmakologie   MeSH
• Listeria monocytogenes * genetika   MeSH
• proteiny vázající GTP genetika   MeSH
• ribozomy genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• linkosamidy MeSH
• proteiny vázající GTP MeSH