Maintaining mitochondrial ribosome function: The role of ribosome rescue and recycling factors.
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ABSTRACT:
SUBMITTER: Nadler F
PROVIDER: S-EPMC8786322 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Characterizing the structural dynamics of the translating ribosome remains a major goal in the study of protein synthesis. Deacylation of peptidyl-tRNA during translation elongation triggers fluctuations of the pretranslocation ribosomal complex between two global conformational states. Elongation factor G-mediated control of the resulting dynamic conformational equilibrium helps to coordinate ribosome and tRNA movements during elongation and is thus a crucial mechanistic feature of translation. Beyond elongation, deacylation of peptidyl-tRNA also occurs during translation termination, and this deacylated tRNA persists during ribosome recycling. Here we report that specific regulation of the analogous conformational equilibrium by translation release and ribosome recycling factors has a critical role in the termination and recycling mechanisms. Our results support the view that specific regulation of the global state of the ribosome is a fundamental characteristic of all translation factors and a unifying theme throughout protein synthesis.
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Project description:RRF (ribosome recycling factor) consists of two domains, and in concert with EF-G (elongation factor-G), triggers dissociation of the post-termination ribosomal complex. However, the function of the individual domains of RRF remains unclear. To clarify this, two RRF chimaeras, EcoDI/TteDII and TteDI/EcoDII, were created by domain swaps between the proteins from Escherichia coli and Thermoanaerobacter tengcongensis. The ribosome recycling activity of the RRF chimaeras was compared with their wild-type RRFs by using in vivo and in vitro activity assays. Like wild-type TteRRF (T. tengcongensis RRF), the EcoDI/TteDII chimaera is non-functional in E. coli, but both wild-type TteRRF, and EcoDI/TteDII can be activated by coexpression of T. tengcongensis EF-G in E. coli. By contrast, like wild-type E. coli RRF (EcoRRF), TteDI/EcoDII is fully functional in E. coli. These findings suggest that domain II of RRF plays a crucial role in the concerted action of RRF and EF-G for the post-termination complex disassembly, and the specific interaction between RRF and EF-G on ribosomes mainly depends on the interaction between domain II of RRF and EF-G. This study provides direct genetic and biochemical evidence for the function of the individual domains of RRF.
| S-EPMC1360730 | biostudies-other
Project description:BackgroundmtRF1 is a vertebrate mitochondrial protein with an unknown function that arose from a duplication of the mitochondrial release factor mtRF1a. To elucidate the function of mtRF1, we determined the positions that are conserved among mtRF1 sequences but that are different in their mtRF1a paralogs. We subsequently modeled the 3D structure of mtRF1a and mtRF1 bound to the ribosome, highlighting the structural implications of these differences to derive a hypothesis for the function of mtRF1.ResultsOur model predicts, in agreement with the experimental data, that the 3D structure of mtRF1a allows it to recognize the stop codons UAA and UAG in the A-site of the ribosome. In contrast, we show that mtRF1 likely can only bind the ribosome when the A-site is devoid of mRNA. Furthermore, while mtRF1a will adopt its catalytic conformation, in which it functions as a peptidyl-tRNA hydrolase in the ribosome, only upon binding of a stop codon in the A-site, mtRF1 appears specifically adapted to assume this extended, peptidyl-tRNA hydrolyzing conformation in the absence of mRNA in the A-site.ConclusionsWe predict that mtRF1 specifically recognizes ribosomes with an empty A-site and is able to function as a peptidyl-tRNA hydrolase in those situations. Stalled ribosomes with empty A-sites that still contain a tRNA bound to a peptide chain can result from the translation of truncated, stop-codon less mRNAs. We hypothesize that mtRF1 recycles such stalled ribosomes, performing a function that is analogous to that of tmRNA in bacteria.
| S-EPMC3418547 | biostudies-literature
Project description:The yeast protein Rrf1p encoded by the FIL1 nuclear gene bears significant sequence similarity to Escherichia coli ribosome recycling factor (RRF). Here, we call FIL1 Ribosome Recycling Factor of yeast, RRF1. Its gene product, Rrf1p, was localized in mitochondria. Deletion of RRF1 leads to a respiratory incompetent phenotype and to instability of the mitochondrial genome (conversion to rho(-)/rho(0) cytoplasmic petites). Yeast with intact mitochondria and with deleted genomic RRF1 that harbors a plasmid carrying RRF1 was prepared from spores of heterozygous diploid yeast. Such yeast with a mutated allele of RRF1, rrf1-L209P, grew on a non-fermentable carbon source at 30 but not at 36 degrees C, where mitochondrial but not total protein synthesis was 90% inhibited. We propose that Rrf1p is essential for mitochondrial protein synthesis and acts as a RRF in mitochondria.
| S-EPMC165964 | biostudies-literature
Project description:Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.
| S-EPMC8209004 | biostudies-literature
Project description:Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans.
| S-EPMC3375252 | biostudies-literature
Project description:The molecular mechanism of human mitochondrial translation has yet to be fully described. We are particularly interested in understanding the process of translational termination and ribosome recycling in the mitochondrion. Several candidates have been implicated, for which subcellular localization and characterization have not been reported. Here, we show that the putative mitochondrial recycling factor, mtRRF, is indeed a mitochondrial protein. Expression of human mtRRF in fission yeast devoid of endogenous mitochondrial recycling factor suppresses the respiratory phenotype. Further, human mtRRF is able to associate with Escherichia coli ribosomes in vitro and can associate with mitoribosomes in vivo. Depletion of mtRRF in human cell lines is lethal, initially causing profound mitochondrial dysmorphism, aggregation of mitoribosomes, elevated mitochondrial superoxide production and eventual loss of OXPHOS complexes. Finally, mtRRF was shown to co-immunoprecipitate a large number of mitoribosomal proteins attached to other mitochondrial proteins, including putative members of the mitochondrial nucleoid.
| S-EPMC2566884 | biostudies-literature
Project description:Mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoribosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoribosome recycling factor (RRFmt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRFmt Here we present a 3.9-Å resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoribosome-RRFmt complex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoribosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRFmt binding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoribosome recycling.
| S-EPMC6486771 | biostudies-literature
Project description:Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.
| S-EPMC7427801 | biostudies-literature
Project description:The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.
| S-EPMC9955659 | biostudies-literature