scholarly journals Structures of the human mitochondrial ribosome recycling complexes reveal distinct mechanisms of recycling and antibiotic resistance

2020 ◽  
Author(s):  
Ravi Kiran Koripella ◽  
Ayush Deep ◽  
Ekansh K. Agrawal ◽  
Pooja Keshavan ◽  
Nilesh K. Banavali ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Conformational Changes ◽  
Messenger Rna ◽  
Elongation Factor ◽  
Ribosome Recycling Factor ◽  
Ribosomal Subunits ◽  
Sequence Of Events ◽  
Mitochondrial Ribosome ◽  
Mitochondrial Elongation

AbstractRibosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Mitochondrial ribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt) are two proteins with mitochondria-specific additional sequences that catalyze the recycling step in human mitochondria. We have determined high-resolution cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) in complex with RRFmt, and the mitoribosomal large 39S subunit in complex with both RRFmt and EF-G2mt. In addition, we have captured the structure of a short-lived intermediate state of the 55S•RRFmt•EF-G2mt complex. These structures clarify the role of a mitochondria-specific segment of RRFmt in mitoribosome recycling, identify the structural distinctions between the two isoforms of EF-Gmt that confer their functional specificity, capture recycling-specific conformational changes in the L7/L12 stalk-base region, and suggest a distinct mechanistic sequence of events in mitoribosome recycling. Furthermore, biochemical and structural assessments of the sensitivity of EF-G2mt to the antibiotic fusidic acid reveals that the molecular mechanism of antibiotic resistance for EF-G2mt is markedly different from that exhibited by mitochondrial elongation factor EF-G1mt, suggesting that these two homologous mitochondrial proteins have evolved diversely to negate the effect of a bacterial antibiotics.

scholarly journals Distinct mechanisms of the human mitoribosome recycling and antibiotic resistance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ravi Kiran Koripella ◽  
Ayush Deep ◽  
Ekansh K. Agrawal ◽  
Pooja Keshavan ◽  
Nilesh K. Banavali ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Elongation Factor ◽  
Acid Resistance ◽  
Functional Specificity ◽  
Ribosomal Subunits ◽  
Sequence Of Events ◽  
Mitochondrial Ribosome ◽  
Distinct Sequence ◽  
Mitochondrial Elongation

AbstractRibosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt). These structures clarify an unusual role of a mitochondria-specific segment of RRFmt, identify the structural distinctions that confer functional specificity to EF-G2mt, and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2mt is markedly different from that of mitochondrial elongation factor EF-G1mt, suggesting that the two human EF-Gmts have evolved diversely to negate the effect of a bacterial antibiotic.

scholarly journals Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dasol Kim ◽  
Hui-Yun Hwang ◽  
Eun Sun Ji ◽  
Jin Young Kim ◽  
Jong Shin Yoo ◽  
...  
Keyword(s):  
Metabolic Regulation ◽  
Elongation Factor ◽  
Dependent Manner ◽  
Diet Induced Obesity ◽  
Metabolic Dysregulation ◽  
Autophagy Induction ◽  
Transcription Factor Eb ◽  
Mitochondrial Elongation

AbstractDisorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

scholarly journals Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

2015 ◽  
Vol 1 (4) ◽  
pp. e1500169 ◽  
Author(s):  
Wen Li ◽  
Zheng Liu ◽  
Ravi Kiran Koripella ◽  
Robert Langlois ◽  
Suparna Sanyal ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Elongation Factor G ◽  
Direct Role ◽  
Gtp Hydrolysis ◽  
Ribosomal Subunits ◽  
Trna Translocation ◽  
Gtpase Activation ◽  
Guanosine Triphosphatase ◽  
Factor G

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

scholarly journals Evaluation on Elongation Factor 1 Alpha of Entamoeba histolytica Interaction with the Intermediate Subunit of the Gal/GalNAc Lectin and Actin in Phagocytosis

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 702
Author(s):  
Hang Zhou ◽  
Yue Guan ◽  
Meng Feng ◽  
Yongfeng Fu ◽  
Hiroshi Tachibana ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Chinese Hamster Ovary ◽  
Messenger Rna ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Elongation Factor 1 Alpha ◽  
Elongation Factor 1 ◽  
Pathogenic Process

Entamoeba histolytica is the causative agent of amoebiasis. This disease results in 40,000 to 100,000 deaths annually. The pathogenic molecules involved in the invasion of trophozoites had been constantly being clarified. This study explored the role of elongation factor 1 alpha (EF1a) in E. histolytica pathogenicity. Biolayer interferometry binding and pull-down assays suggest that EF1a and intermediate subunit of lectin (Igl) binding are specific. Submembranous distribution of EF1a closely aligns with the localization of Igl, which appear in abundance on membranes of trophozoites. Messenger RNA (mRNA) expression of EF1a is positively correlated with trends in Igl levels after co-incubation with Chinese hamster ovary (CHO) cells in vitro, suggesting a regulatory linkage between these proteins. Erythrophagocytosis assays also imply a role for EF1a in phagocytosis. Finally, EF1a and actin are collocated in trophozoites. These results indicated elongation factor 1a is associated with E. histolytica phagocytosis, and the relationships between EF1a, Igl, and actin are worth further study to better understand the pathogenic process.

The post-transcriptional life of mammalian mitochondrial RNA

2012 ◽  
Vol 444 (3) ◽  
pp. 357-373 ◽  
Author(s):  
Joanna Rorbach ◽  
Michal Minczuk
Keyword(s):  
Messenger Rna ◽  
Mitochondrial Gene ◽  
Rna Stability ◽  
Stability Control ◽  
Mitochondrial Rna ◽  
Mammalian Mitochondria ◽  
Mitochondrial Ribosome ◽  
Rna Maturation ◽  
Rna Polyadenylation

Mammalian mitochondria contain their own genome that encodes mRNAs for thirteen essential subunits of the complexes performing oxidative phosporylation as well as the RNA components (two rRNAs and 22 tRNAs) needed for their translation in mitochondria. All RNA species are produced from single polycistronic precursor RNAs, yet the relative concentrations of various RNAs differ significantly. This underscores the essential role of post-transcriptional mechanisms that control the maturation, stability and translation of mitochondrial RNAs. The present review provides a detailed summary on the role of RNA maturation in the regulation of mitochondrial gene expression, focusing mainly on messenger RNA polyadenylation and stability control. Furthermore, the role of mitochondrial ribosomal RNA stability, processing and modifications in the biogenesis of the mitochondrial ribosome is discussed.

scholarly journals Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

1975 ◽  
Vol 67 (1) ◽  
pp. 25-37 ◽  
Author(s):  
B Mechler ◽  
P Vassalli
Keyword(s):  
Protein Synthesis ◽  
Messenger Rna ◽  
Membrane Fraction ◽  
Polypeptide Chain ◽  
Salt Treatment ◽  
Ribosomal Subunits ◽  
Myeloma Cells ◽  
Membrane Bound ◽  
Made In

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.

scholarly journals Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

1982 ◽  
Vol 95 (1) ◽  
pp. 267-277 ◽  
Author(s):  
R J Lapolla ◽  
A M Lambowitz
Keyword(s):  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Ribosomal Subunits ◽  
Mitochondrial Ribosome ◽  
Equilibrium Centrifugation ◽  
Insight Into

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

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