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

2019 ◽  
Author(s):  
Robert Buschauer ◽  
Yoshitaka Matsuo ◽  
Ying-Hsin Chen ◽  
Najwa Alhusaini ◽  
Thomas Sweet ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Specific Interaction ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
A Site ◽  
Sense Codon ◽  
Decoding Efficiency ◽  
Insight Into

Control of 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. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

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

Science ◽  
2020 ◽  
Vol 368 (6488) ◽  
pp. eaay6912 ◽  
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.

scholarly journals Translation elongation and mRNA stability are coupled through the ribosomal A-site

2018 ◽  
Author(s):  
Gavin Hanson ◽  
Najwa Alhusaini ◽  
Nathan Morris ◽  
Thomas Sweet ◽  
Jeff Coller
Keyword(s):  
Amino Acid ◽  
Mrna Stability ◽  
Transcript Level ◽  
Amino Acid Identity ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Translation Elongation ◽  
Acid Identity ◽  
Optimal Codons ◽  
A Site

AbstractMessenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in eukaryotic cells. Previous work by us and others has shown that codon identity exerts a powerful influence on mRNA stability. In Saccharomyces cerevisiae, studies using a handful of reporter mRNAs show that optimal codons increase translation elongation rate, which in turn increase mRNA stability. However, a direct link between elongation rate and mRNA stability has not been established across the entire yeast transcriptome. In addition, there is evidence from work in higher eukaryotes that amino acid identity influences mRNA stability, raising the question as to whether the impact of translation elongation on mRNA decay is at the level of tRNA decoding, amino acid incorporation, or some combination of each. To address these questions, we performed ribosome profiling of wildtype yeast. In good agreement with other studies, our data showed faster codon-specific elongation over optimal codons and faster transcript-level elongation correlating with transcript optimality. At both the codon-level and transcript-level, faster elongation correlated with increased mRNA stability. These findings were reinforced by showing increased translation efficiency and kinetics for a panel of 11 HIS3 reporter mRNAs of increasing codon optimality. While we did observe that elongation measured by ribosome profiling is composed of both amino acid identity and synonymous codon effects, further analyses of these data establish that A-site tRNA decoding rather than other steps of translation elongation is driving mRNA decay in yeast.

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

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.

scholarly journals Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

2021 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Attilio Fabbretti ◽  
Pohl Milon ◽  
Xu Han ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Resistance Mechanisms ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
A Site ◽  
Insight Into ◽  
Major Target

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

scholarly journals Structure of Gcn1 bound to stalled and colliding 80S ribosomes

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.

scholarly journals Anatomy of footprint extension in ribosome profiling reveals translational landscape mediated by S1 protein in bacteria

2020 ◽  
Author(s):  
Tomoya Fujita ◽  
Takeshi Yokoyama ◽  
Mikako Shirouzu ◽  
Hideki Taguchi ◽  
Takuhiro Ito ◽  
...  
Keyword(s):  
Rna Secondary Structure ◽  
Rna Binding ◽  
Ribosome Profiling ◽  
Powerful Method ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
Bacterial Ribosome ◽  
Conformational Diversity ◽  
Rna Duplexes ◽  
Rnase Digestion

Abstract Ribosome profiling — RNase footprinting of ribosome-bound mRNA — has been a unique and powerful method, applied to widespread organisms to survey ribosome traversal along mRNAs. In contrast to eukaryotes, bacterial ribosome footprints show a broad range of sizes, reflecting the differential states of ribosomes. However, the origin remains unclear. Here, we show that rotated state of ribosomes and intramolecular RNA duplexes each extend bacterial ribosome footprints at the 5′ end. Combining elongation inhibitors, cryo-electron microscopy, and ribosome profiling, we demonstrated that the rotated state of ribosomes results in long footprints. Along the subunit rotation, ribosomal protein S1 — a 30S-subunit RNA-binding protein — sterically protects mRNA at the 5′ end of the ribosome from RNase digestion and facilitates elongation of the ribosome. Moreover, we found that ribosomes stalled on ycbZ mRNA generate prolonged footprints because of their unique RNA secondary structure proximal to ribosomes. Through the studies of footprint extension, our results revealed S1-mediated stabilization of translation elongation and provide ribosome profiling approach to probe the conformational diversity of ribosomes in bacteria.

scholarly journals Disome-seq reveals widespread ribosome collisions that recruit co-translational chaperones

2019 ◽  
Author(s):  
Taolan Zhao ◽  
Yan-Ming Chen ◽  
Yu Li ◽  
Jia Wang ◽  
Siyu Chen ◽  
...  
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Protein Homeostasis ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Protein Levels ◽  
Cryo Electron Microscopy ◽  
Exit Tunnel ◽  
Hidden Layer ◽  
A Site

ABSTRACTRegulation of translation elongation plays a crucial role in determining absolute protein levels and ensuring the correct localization and folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes (ribo-seq). However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. 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′-ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are missed in traditional ribo-seq. These collisions are due to 1) slow ribosome release when stop codons are at the A-site, 2) slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. The paused ribosomes can continue translating after collisions, as suggested by the structure of disomes obtained by cryo-electron microscopy (cryo-EM). Collided ribosomes recruit chaperones, which can aid in the co-translational folding of the nascent peptides. Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

scholarly journals Translation elongation and mRNA stability are coupled through the ribosomal A-site

RNA ◽  
2018 ◽  
Vol 24 (10) ◽  
pp. 1377-1389 ◽  
Author(s):  
Gavin Hanson ◽  
Najwa Alhusaini ◽  
Nathan Morris ◽  
Thomas Sweet ◽  
Jeff Coller
Keyword(s):  
Mrna Stability ◽  
Translation Elongation ◽  
A Site

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

2020 ◽  
Vol 117 (17) ◽  
pp. 9630-9641 ◽  
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.

scholarly journals The Halastavi árva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

2020 ◽  
Author(s):  
Irina S. Abaeva ◽  
Quentin Vicens ◽  
Anthony Bochler ◽  
Heddy Soufari ◽  
Angelita Simonetti ◽  
...  
Keyword(s):  
Intergenic Region ◽  
Internal Ribosomal Entry Sites ◽  
Initiation Mechanism ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Elongation Cycle ◽  
Cryo Electron Microscopy ◽  
A Site ◽  
Sense Codon

ABSTRACTDicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi árva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

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