Structure of the translating Neurospora ribosome arrested by cycloheximide

Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX’s mechanism of action based on the cryo-electron microscopy structure of actively translating Neurospora crassa ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from Neurospora, yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating N. crassa ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.

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The third-generation tetracycline, KBP-7072, exploits and reveals a new potential of the primary tetracycline binding pocket

10.1101/508218 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuya Kaminishi ◽

Andreas Schedlbauer ◽

Borja Ochoa-Lizarralde ◽

Elisa de Astigarraga ◽

Retina Çapuni ◽

...

Keyword(s):

Antibiotic Resistance ◽

Ribosomal Subunit ◽

Binding Pocket ◽

Protein Synthesis Inhibitor ◽

Third Generation ◽

Bacterial Strains ◽

Synthesis Inhibitor ◽

The Third ◽

Spectrum Activity ◽

A Site

Antibiotic resistance is a growing threat to human health requiring the discovery or development of new anti-infectives. As such, KBP-7072 is a novel tetracycline derivative that exhibits broad-spectrum activity against Gram-positive and -negative bacterial strains. To determine the mechanism of action of KBP-7072 and understand how its unique C9 extension can be used to combat the growing problem of antibiotic resistance we determined the structure of KBP-7072 bound to the bacterial 30S ribosomal subunit, the inhibitory target of typical tetracyclines. We show that KBP-7072 binds to the primary tetracycline binding site on the 30S ribosomal subunit consistent with it acting as a protein synthesis inhibitor that blocks A-site occupation. Moreover, the unique chemical nature of KBP-7072s C9 extension leads to a distinctive interaction pattern with the 30S subunit that distinguishes KBP-7072 from the third-generation tetracycline, Tigecycline, and thus expands the interaction potential of the primary tetracycline binding pocket.

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Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The Journal of Cell Biology ◽

10.1083/jcb.201001124 ◽

2010 ◽

Vol 189 (7) ◽

pp. 1079-1086 ◽

Cited By ~ 49

Author(s):

Jayati Sengupta ◽

Cyril Bussiere ◽

Jesper Pallesen ◽

Matthew West ◽

Arlen W. Johnson ◽

...

Keyword(s):

Binding Site ◽

Nuclear Export ◽

Large Subunit ◽

Ribosomal Subunit ◽

Adaptor Protein ◽

Structural Data ◽

Release Mechanism ◽

Nuclear Pore ◽

Subunit Joining

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

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The Ccr4-Not complex monitors the translating ribosome for codon optimality

Science ◽

10.1126/science.aay6912 ◽

2020 ◽

Vol 368 (6488) ◽

pp. eaay6912 ◽

Cited By ~ 22

Author(s):

Robert Buschauer ◽

Yoshitaka Matsuo ◽

Takato Sugiyama ◽

Ying-Hsin Chen ◽

Najwa Alhusaini ◽

...

Keyword(s):

Mrna Stability ◽

Messenger Rna ◽

Mrna Decay ◽

Specific Interaction ◽

Ribosome Profiling ◽

Translation Elongation ◽

Cryo Electron Microscopy ◽

A Site ◽

Sense Codon ◽

Decoding Efficiency

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo–electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae. This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.

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High affinity promoter binding of STOP1 is essential for the early aluminum-inducible expression of novel Al resistance genes GDH1 and GDH2 in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/erab031 ◽

2021 ◽

Author(s):

Mutsutomo Tokizawa ◽

Takuo Enomoto ◽

Hiroki Ito ◽

Liujie Wu ◽

Yuriko Kobayashi ◽

...

Keyword(s):

Binding Site ◽

Resistance Genes ◽

Translational Regulation ◽

Molecular Mechanisms ◽

Aluminum Toxicity ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Induced Expression ◽

Aluminum Resistance ◽

High Affinity

Abstract Malate-efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1), which mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum-resistance in Arabidopsis thaliana. Several studies showed that root AtALMT1 expression is rapidly observed in response to aluminum (within 1-hour), this early induction is an important mechanism to immediately protect roots from aluminum-toxicity. Additionally, identifying the molecular mechanisms that underlie rapid aluminum-resistance responses should lead to a better understanding of plant aluminum-sensing and -signal transduction mechanisms. In this study, histochemical analyses using GFP-tagged STOP1 proteins showed that STOP1 proteins were accumulated in the nucleus soon after aluminum-treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1-induction were observed in the presence of the protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2), but not for Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for the early induction. Finally, we report that GDH1 and 2 which are the target of STOP1 are novel aluminum-resistance genes in Arabidopsis.

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Joining the interface: a site for Nmd3 association on 60S ribosome subunits

The Journal of Cell Biology ◽

10.1083/jcb.201006033 ◽

2010 ◽

Vol 189 (7) ◽

pp. 1071-1073 ◽

Cited By ~ 2

Author(s):

Marlene Oeffinger

Keyword(s):

Binding Site ◽

Ribosome Biogenesis ◽

Large Subunit ◽

Adaptor Protein ◽

Cryoelectron Microscopy ◽

Structural Description ◽

Ribosomal Subunits ◽

Cell Biol ◽

Conformational Model ◽

A Site

The adaptor protein Nmd3 is required for Crm1-dependent export of large ribosomal subunits from the nucleus. In this issue, Sengupta et al. (2010. J. Cell Biol. doi:10.1083/jcb.201001124) identify a binding site for yeast Nmd3 on 60S ribosomal subunits using cryoelectron microscopy and suggest a conformational model for its release in the cytoplasm. The study provides the first detailed structural description of a ribosome biogenesis factor in complex with the large subunit.

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Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1918145117 ◽

2020 ◽

Vol 117 (17) ◽

pp. 9630-9641 ◽

Cited By ~ 2

Author(s):

Cédric Gobet ◽

Benjamin Dieter Weger ◽

Julien Marquis ◽

Eva Martin ◽

Nagammal Neelagandan ◽

...

Keyword(s):

Codon Usage ◽

Messenger Rna ◽

Mouse Liver ◽

Ribosome Profiling ◽

Translation Elongation ◽

Speed Up ◽

Codon Pair ◽

Feeding Regimen ◽

Higher Eukaryotes ◽

A Site

Translation depends on messenger RNA (mRNA)-specific initiation, elongation, and termination rates. While translation elongation is well studied in bacteria and yeast, less is known in higher eukaryotes. Here we combined ribosome and transfer RNA (tRNA) profiling to investigate the relations between translation elongation rates, (aminoacyl-) tRNA levels, and codon usage in mammals. We modeled codon-specific ribosome dwell times from ribosome profiling, considering codon pair interactions between ribosome sites. In mouse liver, the model revealed site- and codon-specific dwell times that differed from those in yeast, as well as pairs of adjacent codons in the P and A site that markedly slow down or speed up elongation. While translation efficiencies vary across diurnal time and feeding regimen, codon dwell times were highly stable and conserved in human. Measured tRNA levels correlated with codon usage and several tRNAs showed reduced aminoacylation, which was conserved in fasted mice. Finally, we uncovered that the longest codon dwell times could be explained by aminoacylation levels or high codon usage relative to tRNA abundance.

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Isolation and partial characterization of a protein synthesis inhibitor from brine shrimp embryos

Canadian Journal of Biochemistry ◽

10.1139/o77-144 ◽

1977 ◽

Vol 55 (9) ◽

pp. 965-974 ◽

Cited By ~ 7

Author(s):

A. H. Warner ◽

V. Shridhar ◽

F. J. Finamore

Keyword(s):

Protein Synthesis ◽

Translational Control ◽

Brine Shrimp ◽

Elongation Factor ◽

Artemia Salina ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Elongation Step ◽

Artemia Cysts ◽

Inhibitor Of Protein Synthesis

Encysted embryos of the brine shrimp, Artemia salina, contain an inhibitor of protein synthesis that appears to be important in translational control. In cyst homogenates, the inhibitor appears to be partitioned almost equally between the cytosol and ribosome fractions and it has been purified from both fractions to near homogeneity. In a cell-free protein-synthesizing system derived from Artemia cysts, with poly(U) as messenger, the protein inhibits polyphenylalanine synthesis proportional to inhibitor concentration up to about 75% inhibition, and the primary site of action appears to be at the elongation step. The inhibitor activity is not altered by 50–150 mM KCl in the reaction mixture, but it is slightly more effective at 5 mM MgCl2 than at 10 mM MgCl2. The inhibitor is a heat-labile protein of 130 000 molecular weight and is devoid of hydrolase activity. Our data indicate that the inhibitor is not elongation factor EF-1 or EF-2, but we are studying the possibility that it may be a modified form of elongation factor EF-2.

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Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661566 ◽

1986 ◽

Vol 55 (03) ◽

pp. 369-374 ◽

Cited By ~ 15

Author(s):

Raffaele De Caterina ◽

Babette B Weksler

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Arachidonic Acid Metabolism ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Prolonged Incubation ◽

Dependent Inhibition ◽

Human Thrombin ◽

Umbilical Vein Endothelial Cells

SummaryTo learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.

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