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The Universally Conserved ATPase YchF Regulates Translation of Leaderless mRNA in Response to Stress Conditions.

ABSTRACT: The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, and intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promoter fusion experiments, we demonstrate that YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress, or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor 3 (IF3) and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs, and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation.

SUBMITTER: Landwehr V

PROVIDER: S-EPMC8138138 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The universally conserved ATPase YchF regulates translation of leaderless mRNA in response to stress conditions

Project description:The universally conserved P-loop GTPases control diverse cellular processes, like signal transduction, ribosome assembly, cell motility, intracellular transport and translation. YchF belongs to the Obg-family of P-loop GTPases and is one of the least characterized member of this family. It is unique because it preferentially hydrolyses ATP rather than GTP, but its physiological role is largely unknown. Studies in different organisms including humans suggest a possible role of YchF in regulating the cellular adaptation to stress conditions. In the current study, we explored the role of YchF in the model organism Escherichia coli. By western blot and promotor fusion experiments, we demonstrate that the YchF levels decrease during stress conditions or when cells enter stationary phase. The decline in YchF levels trigger increased stress resistance and cells lacking YchF are resistant to multiple stress conditions, like oxidative stress, replication stress or translational stress. By in vivo site directed cross-linking we demonstrate that YchF interacts with the translation initiation factor IF3 and with multiple ribosomal proteins at the surface of the small ribosomal subunit. The absence of YchF enhances the anti-association activity of IF3, stimulates the translation of leaderless mRNAs and increases the resistance against the endoribonuclease MazF, which generates leaderless mRNAs during stress conditions. In summary, our data identify YchF as a stress-responsive regulator of leaderless mRNA translation

2021-09-09 | PXD023192 | Pride

The Role of the Universally Conserved ATPase YchF/Ola1 in Translation Regulation during Cellular Stress.

Project description:The ability to respond to metabolic or environmental changes is an essential feature in all cells and involves both transcriptional and translational regulators that adjust the metabolic activity to fluctuating conditions. While transcriptional regulation has been studied in detail, the important role of the ribosome as an additional player in regulating gene expression is only beginning to emerge. Ribosome-interacting proteins are central to this translational regulation and include universally conserved ribosome interacting proteins, such as the ATPase YchF (Ola1 in eukaryotes). In both eukaryotes and bacteria, the cellular concentrations of YchF/Ola1 determine the ability to cope with different stress conditions and are linked to several pathologies in humans. The available data indicate that YchF/Ola1 regulates the stress response via controlling non-canonical translation initiation and via protein degradation. Although the molecular mechanisms appear to be different between bacteria and eukaryotes, increased non-canonical translation initiation is a common consequence of YchF/Ola1 regulated translational control in E. coli and H. sapiens. In this review, we summarize recent insights into the role of the universally conserved ATPase YchF/Ola1 in adapting translation to unfavourable conditions.

| S-EPMC8779481 | biostudies-literature

Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1.

Project description:AimsYchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response.ResultsOur data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA.InnovationYchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif.ConclusionOur data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.

| S-EPMC4742990 | biostudies-literature

Histidine 114 Is Critical for ATP Hydrolysis by the Universally Conserved ATPase YchF.

Project description:GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family.

| S-EPMC4513122 | biostudies-literature

A universally conserved ATPase regulates the oxidative stress response in Escherichia coli.

Project description:YchF is an evolutionarily conserved ATPase of unknown function. In humans, the YchF homologue hOla1 appears to influence cell proliferation and was found to be up-regulated in many tumors. A possible involvement in regulating the oxidative stress response was also suggested, but details on the underlying mechanism are lacking. For gaining insight into YchF function, we used Escherichia coli as a model organism and found that YchF overexpression resulted in H(2)O(2) hypersensitivity. This was not caused by transcriptional or translational down-regulation of H(2)O(2)-scavenging enzymes. Instead, we observed YchF-dependent inhibition of catalase activity and a direct interaction with the major E. coli catalase KatG. KatG inhibition was dependent on the ATPase activity of YchF and was regulated by post-translational modifications, most likely including an H(2)O(2)-dependent dephosphorylation. We furthermore showed that YchF expression is repressed by the transcription factor OxyR and further post-translationally modified in response to H(2)O(2). In summary, our data show that YchF functions as a novel negative regulator of the oxidative stress response in E. coli. Considering the available data on hOla1, YchF/Ola1 most likely execute similar functions in bacteria and humans, and their up-regulation inhibits the ability of the cells to scavenge damaging reactive oxygen species.

| S-EPMC3527945 | biostudies-literature

The Universally Conserved Unconventional G Protein YchF Is Critical for Growth and Stress Response.

Project description:The ancient guanine nucleotide-binding (G) proteins are a group of critical regulatory and signal transduction proteins, widely involved in diverse cellular processes of all kingdoms of life. YchF is a kind of universally conserved novel unconventional G protein that appears to be crucial for growth and stress response in eukaryotes and bacteria. YchF is able to bind and hydrolyze both adenine nucleoside triphosphate (ATP) and guanosine nucleoside triphosphate (GTP), unlike other members of the P-loop GTPases. Hence, it can transduce signals and mediate multiple biological functions by using either ATP or GTP. YchF is not only a nucleotide-dependent translational factor associated with the ribosomal particles and proteasomal subunits, potentially bridging protein biosynthesis and degradation, but also sensitive to reactive oxygen species (ROS), probably recruiting many partner proteins in response to environmental stress. In this review, we summarize the latest insights into how YchF is associated with protein translation and ubiquitin-dependent protein degradation to regulate growth and maintain proteostasis under stress conditions.

| S-EPMC10144078 | biostudies-literature

Universally conserved translation initiation factors.

Project description:The process by which translation is initiated has long been considered similar in Bacteria and Eukarya but accomplished by a different unrelated set of factors in the two cases. This not only implies separate evolutionary histories for the two but also implies that at the universal ancestor stage, a translation initiation mechanism either did not exist or was of a different nature than the extant processes. We demonstrate herein that (i) the "analogous" translation initiation factors IF-1 and eIF-1A are actually related in sequence, (ii) the "eukaryotic" translation factor SUI1 is universal in distribution, and (iii) the eukaryotic/archaeal translation factor eIF-5A is homologous to the bacterial translation factor EF-P. Thus, the rudiments of translation initiation would seem to have been present in the universal ancestor stage. However, significant development and refinement subsequently occurred independently on both the bacterial lineage and on the archaeal/eukaryotic line.

| S-EPMC18182 | biostudies-literature

Translation of the leaderless Caulobacter dnaX mRNA.

Project description:The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

| S-EPMC179208 | biostudies-other

A combination of mRNA features influence the efficiency of leaderless mRNA translation initiation.

Project description:Bacterial translation is thought to initiate by base pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNA's 5' untranslated region (UTR). However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5' UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using these data, a simple computational model was built based on the combinatorial relationship of these mRNA features that can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

| S-EPMC8459731 | biostudies-literature

Four translation initiation pathways employed by the leaderless mRNA in eukaryotes.

Project description:mRNAs lacking 5' untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.

| S-EPMC5124965 | biostudies-literature

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