Structure of Gcn1 bound to stalled and colliding 80S ribosomes

AbstractProtein synthesis is essential to cells and requires a constant supply of nutrients. Amino acid starvation leads to accumulation of uncharged tRNAs that promote ribosomal stalling, which is sensed by the protein kinase Gcn2, together with its effector proteins, Gcn1 and Gcn20. Activation of Gcn2 phosphorylates eIF2, leading to a global repression of translation. Fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, which is a negative regulator of Gcn2. Despite the wealth of biochemical data, structures of Gcn proteins on the ribosome have remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the A-site region of the lead ribosome. The lead ribosome is stalled in a non-rotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, as well as Rbg2/Gir2 located in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings provide a structural basis for Rbg2/Gir2 repression of Gcn2, and also reveal that colliding disomes are the substrate for Gcn1 binding, which has important implications not only for Gcn2-activated stress responses, but also for general ribosome quality control (RQC) pathways.

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Structure of Gcn1 bound to stalled and colliding 80S ribosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2022756118 ◽

2021 ◽

Vol 118 (14) ◽

pp. e2022756118

Author(s):

Agnieszka A. Pochopien ◽

Bertrand Beckert ◽

Sergo Kasvandik ◽

Otto Berninghausen ◽

Roland Beckmann ◽

...

Keyword(s):

Negative Regulator ◽

Fine Tuning ◽

Effector Protein ◽

Binding Region ◽

Site Factor ◽

Cryo Electron Microscopy ◽

Interaction Mode ◽

A Site ◽

Crucial Part ◽

Insight Into

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

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SPX-related genes regulate phosphorus homeostasis in the marine phytoplankton, Phaeodactylum tricornutum

Communications Biology ◽

10.1038/s42003-021-02284-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Kaidian Zhang ◽

Zhi Zhou ◽

Jiashun Li ◽

Jingtian Wang ◽

Liying Yu ◽

...

Keyword(s):

Stress Responses ◽

Phaeodactylum Tricornutum ◽

Response Regulator ◽

P Uptake ◽

Negative Regulator ◽

Marine Phytoplankton ◽

Alkaline Phosphatases ◽

Phosphate Starvation Response ◽

Essential Nutrient ◽

Global Oceans

AbstractPhosphorus (P) is an essential nutrient for marine phytoplankton. Maintaining intracellular P homeostasis against environmental P variability is critical for phytoplankton, but how they achieve this is poorly understood. Here we identify a SPX gene and investigate its role in Phaeodactylum tricornutum. SPX knockout led to significant increases in the expression of phosphate transporters, alkaline phosphatases (the P acquisition machinery) and phospholipid hydrolases (a mechanism to reduce P demand). These demonstrate that SPX is a negative regulator of both P uptake and P-stress responses. Furthermore, we show that SPX regulation of P uptake and metabolism involves a phosphate starvation response regulator (PHR) as an intermediate. Additionally, we find the SPX related genes exist and operate across the phytoplankton phylogenetic spectrum and in the global oceans, indicating its universal importance in marine phytoplankton. This study lays a foundation for better understanding phytoplankton adaptation to P variability in the future changing oceans.

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Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

Genome Biology ◽

10.1186/s13059-020-02256-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Taolan Zhao ◽

Yan-Ming Chen ◽

Yu Li ◽

Jia Wang ◽

Siyu Chen ◽

...

Keyword(s):

Protein Quality ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Translation Elongation ◽

Cryo Electron Microscopy ◽

Cotranslational Folding ◽

A Cell ◽

Exit Tunnel ◽

Hidden Layer ◽

A Site

Abstract Background The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. Results In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. Conclusions Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

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Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies

Science Advances ◽

10.1126/sciadv.abd4413 ◽

2021 ◽

Vol 7 (2) ◽

pp. eabd4413

Author(s):

Jung-Hoon Lee ◽

Daniel Bollschweiler ◽

Tillman Schäfer ◽

Robert Huber

Keyword(s):

Transcriptional Repression ◽

Histone Deacetylases ◽

Active Sites ◽

Chromatin Modification ◽

Structural Basis ◽

Amino Terminal ◽

Cryo Electron Microscopy ◽

Multiple Contacts ◽

Lysine Residues ◽

Nucleosome Binding

The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from acetyl-lysine residues in histone amino-terminal tails, thereby mediating transcriptional repression. Structural makeup and mechanisms by which multisubunit HDAC complexes recognize nucleosomes remain elusive. Our cryo–electron microscopy structures of the yeast class II HDAC ensembles show that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely accessible. We also observe a tetramer of protomers, where the nucleosome binding modules are inaccessible. Structural analysis of the nucleosome-bound complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC catalysis. Moreover, it reveals how an intricate network of multiple contacts between a dimer of protomers and the nucleosome creates a platform for expansion of the HDAC activities. Our study provides comprehensive insight into the structural plasticity of the HDAC complex and its functional mechanism of chromatin modification.

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The Arabidopsis mediator complex subunit 8 regulates oxidative stress responses

The Plant Cell ◽

10.1093/plcell/koab079 ◽

2021 ◽

Cited By ~ 1

Author(s):

Huaming He ◽

Jordi Denecker ◽

Katrien Van Der Kelen ◽

Patrick Willems ◽

Robin Pottie ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Salicylic Acid ◽

Jasmonic Acid ◽

Stress Responses ◽

Negative Regulator ◽

Mediator Complex ◽

Defense Gene Expression ◽

A Genome ◽

Oxidative Stress Responses

Abstract Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.

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Significance of bedrock as a site factor for Aleppo pine

Forest Ecology and Management ◽

10.1016/0378-1127(82)90001-9 ◽

1982 ◽

Vol 4 (3) ◽

pp. 213-223 ◽

Cited By ~ 17

Author(s):

G. Schiller

Keyword(s):

Aleppo Pine ◽

Site Factor ◽

A Site

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Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽

10.7554/elife.22153 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 94

Author(s):

Akira Karasawa ◽

Toshimitsu Kawate

Keyword(s):

P2x7 Receptor ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Drug Binding ◽

Structural Basis ◽

Atp Binding ◽

Allosteric Site ◽

Channel Opening ◽

A Site ◽

Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

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Structural insights into the activation of human calcium-sensing receptor

10.1101/2021.03.30.437720 ◽

2021 ◽

Author(s):

Xiaochen Chen ◽

Lu Wang ◽

Zhanyu Ding ◽

Qianqian Cui ◽

Li Han ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Transmembrane Domains ◽

Structural Basis ◽

Calcium Sensing Receptor ◽

Calcium Sensing ◽

Cryo Electron Microscopy ◽

Activation Mechanisms ◽

G Protein Coupled ◽

Structural Insights

AbstractHuman calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and active states. Complemented with previously reported crystal structures of CaSR extracellular domains, it suggests that there are three distinct conformations: inactive, intermediate and active state during the activation. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data shows that the agonist binding leads to the compaction of the dimer, the proximity of the cysteine-rich domains, the large-scale transitions of 7-transmembrane domains, and the inter-and intrasubunit conformational changes of 7-transmembrane domains to accommodate the downstream transducers. Our results reveal the structural basis for activation mechanisms of the CaSR.

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Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Science ◽

10.1126/science.1259724 ◽

2015 ◽

Vol 347 (6217) ◽

pp. 75-78 ◽

Cited By ~ 154

Author(s):

Peter S. Shen ◽

Joseph Park ◽

Yidan Qin ◽

Xueming Li ◽

Krishna Parsawar ◽

...

Keyword(s):

Electron Microscopy ◽

Transfer Rna ◽

Exit Channel ◽

Ribosomal Subunits ◽

Cryo Electron Microscopy ◽

Nascent Chain ◽

New Gene ◽

Carboxy Terminal ◽

A Site ◽

Control Complex

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

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