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ribosome stall

Nathan Zuzow, Arit Ghosh, Marilyn Leonard, Jeffrey Liao, Bing Yang, Eric J Bennett
Previous genetic and biochemical studies from S. cerevisiae have identified a critical ribosome-associated quality control complex (RQC) that facilitates resolution of stalled ribosomal complexes. While components of the mammalian RQC have been examined in vitro , a systematic characterization of RQC protein interactions in mammalian cells has yet to be described. Here, we utilize both proximity-labeling proteomic approaches, BioID and APEX, and traditional affinity-based strategies to both identify interacting proteins of mammalian RQC members and putative substrates for the RQC resident E3 ligase, Ltn1...
March 14, 2018: Molecular Biology of the Cell
Widler Casy, Austin R Prater, Peter V Cornish
During translation, the small subunit of the ribosome rotates with respect to the large subunit primarily between two states as mRNA is being translated into a protein. At the termination of bacterial translation, class I release factors (RFs) bind to a stop codon in the A-site and catalyze the release of the peptide chain from the ribosome. Periodically, mRNA is truncated prematurely and the translating ribosome stalls at the end of the mRNA forming a non-stop complex requiring one of several ribosome rescue factors to intervene...
March 2, 2018: Biochemistry
René Toribio, Irene Díaz-López, Jasminka Boskovic, Iván Ventoso
The topology and dynamics of the scanning ribosomal 43S pre-initiation complex (PIC) bound to mRNA and initiation factors (eIFs) are probably the least understood aspects of translation initiation in eukaryotes. Recently, we described a trapping mechanism in alphavirus that stalls the PIC during scanning of viral mRNA. Using this model, we were able to snapshot for the first time the eIF4A helicase bound to mRNA in a 48S initiation complex assembled in vitro. This interaction was only detected in the presence of the natural stem loop structure (DLP) located downstream from the AUG in viral mRNA that promoted stalling of the PIC, suggesting that DLP stability was enough to jam the helicase activity of eIF4A in a fraction of assembled 48S complexes...
February 5, 2018: Nucleic Acids Research
Fei Qi, Magdalena Motz, Kirsten Jung, Jürgen Lassak, Dmitrij Frishman
Translation of consecutive prolines causes ribosome stalling, which is alleviated but cannot be fully compensated by the elongation factor P. However, the presence of polyproline motifs in about one third of the E. coli proteins underlines their potential functional importance, which remains largely unexplored. We conducted an evolutionary analysis of polyproline motifs in the proteomes of 43 E. coli strains and found evidence of evolutionary selection against translational stalling, which is especially pronounced in proteins with high translational efficiency...
February 1, 2018: PLoS Computational Biology
Brittany D Bennett, Kaitlyn E Redford, Jeffrey A Gralnick
Shewanella oneidensis strain MR-1 is a versatile bacterium capable of respiring extracellular, insoluble ferric oxide minerals under anaerobic conditions. The respiration of iron minerals results in the production of soluble ferrous ions, which at high concentrations are toxic to living organisms. It is not fully understood how Fe2+ is toxic to cells anaerobically; nor is it fully understood how S. oneidensis is able to resist high levels of Fe2+ Here we describe the results of a transposon-mutant screen and subsequent deletion of the genes clpX and clpP in S...
January 29, 2018: Journal of Bacteriology
Misaki Ishizuka, Yu Imai, Keiichiro Mukai, Kazuma Shimono, Ryoko Hamauzu, Kozo Ochi, Takeshi Hosaka
Lincomycin forms cross-links within the peptidyl transferase loop region of the 23S ribosomal RNA (rRNA) of the 50S subunit of the bacterial ribosome, which is the site of peptide bond formation, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains, suggesting that activation of these strains by utilizing the dose-dependent response of lincomycin could be used to effectively induce the production of cryptic secondary metabolites...
January 25, 2018: Antonie Van Leeuwenhoek
Raphael J Morscher, Gregory S Ducker, Sophia Hsin-Jung Li, Johannes A Mayer, Zemer Gitai, Wolfgang Sperl, Joshua D Rabinowitz
Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored...
January 24, 2018: Nature
Xiao P Peng, Shelly Lim, Shibai Li, Lisette Marjavaara, Andrei Chabes, Xiaolan Zhao
Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein...
January 2018: PLoS Genetics
Qing Feng, Sichen Shao
Protein translation is tightly regulated to ensure high-fidelity expression of genetic information. Various conditions cause ribosomes to stall while synthesizing new proteins. Different types of translational arrest initiate specific mRNA surveillance, protein quality control, and stress response pathways that directly impact gene expression and protein homeostasis. Our understanding of these pathways is greatly enhanced by reconstituting these processes in cell-free systems. The high degree of biochemical manipulability of in vitro systems facilitates the identification of key machineries, mechanistic dissection of their functional roles, and structural analysis of intermediate complexes...
December 22, 2017: Methods: a Companion to Methods in Enzymology
Ryan M Jamiolkowski, Chunlai Chen, Barry S Cooperman, Yale E Goldman
The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation...
December 5, 2017: Biophysical Journal
Jing Shi, Yiwei Liu, Yueying Zhang, Yongxin Jin, Fang Bai, Zhihui Cheng, Shouguang Jin, Weihui Wu
Pseudomonas aeruginosa causes various acute and chronic infections in humans. Treatment with azithromycin (AZM) has been shown to benefit patients with chronic P. aeruginosa infections. By binding to the exit tunnel of the 50S ribosome, AZM causes ribosome stalling and depletion of the intracellular tRNA pool. It has been shown that AZM is able to kill stationary-phase P. aeruginosa cells and repress quorum sensing-regulated virulence factors as well as swarming motility. In P. aeruginosa , the PA5470 gene encodes a putative peptide chain release factor whose expression is highly induced by macrolide antibiotics...
February 2018: Antimicrobial Agents and Chemotherapy
Stefan G Kreft, Elke Deuerling
In eukaryotes, a cytosolic ribosome quality control complex recycles erroneously stalled ribosomes and modifies faulty nascent chains by ubiquitination and by C-terminal Ala- and Thr-extension (CAT-tailing). Reported recently in Cell, Izawa et al. identify cytosolic Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive 1) as an inhibitor of CAT-tailing, which prevents mitochondrial dysfunction caused by imported CAT-tailed polypeptides.
January 2018: Trends in Cell Biology
Isaia Barbieri, Konstantinos Tzelepis, Luca Pandolfini, Junwei Shi, Gonzalo Millán-Zambrano, Samuel C Robson, Demetrios Aspris, Valentina Migliori, Andrew J Bannister, Namshik Han, Etienne De Braekeleer, Hannes Ponstingl, Alan Hendrick, Christopher R Vakoc, George S Vassiliou, Tony Kouzarides
N6 -methyladenosine (m6 A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice...
December 7, 2017: Nature
Jean-Clement Mars, Marianne Sabourin-Felix, Michel G Tremblay, Tom Moss
The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein-DNA interaction profiles. Here we describe a simple numerical deconvolution approach that in large part corrects for this variability and significantly improves both the resolution and quantitation of protein-DNA interaction maps deduced from ChIP-Seq data...
November 20, 2017: G3: Genes—Genomes—Genetics
Takayuki Katoh, Yoshihiko Iwane, Hiroaki Suga
A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA's D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid...
November 16, 2017: Nucleic Acids Research
Youjin Jung, Hag Dong Kim, Hee Woong Yang, Hye Jin Kim, Chang-Young Jang, Joon Kim
When a ribosome complex is stalled during the translation elongation process in eukaryotes, the mono-ubiquitination of Rps3 has recently been shown to be critical to ribosome quality control. We have discovered that the regulatory role of Rps3 mono-ubiquitination is controlled by a deubiquitinase. We also showed that an autophagic signal appears to be coupled to the mono-ubiquitination of Rps3p through the entrance of Ubp3p into the autophagosome in yeasts. The mono-ubiquitination of the Rps3 protein is tightly modulated by reciprocal action between the Hel2p E3 ligase and the Ubp3p deubiquitinase in yeasts and the reciprocal action between the RNF123 E3 ligase and the USP10 deubiquitinase in mammalian cells...
November 17, 2017: Experimental & Molecular Medicine
Tatsuo Serikawa, Christos Spanos, Annekathrin von Hacht, Nediljko Budisa, Juri Rappsilber, Jens Kurreck
G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry...
November 9, 2017: Biochimie
Lilian Lamech, Cole M Haynes
Ribosome stalling results in the production of truncated proteins that can cause proteotoxic stress if not efficiently degraded. A recent paper by Izawa et al. (2017) identifies Vms1 as a key player in the regulation of ribosome quality control specifically on mitochondria-localized ribosomes, ultimately preventing protein aggregate accumulation within mitochondria.
November 6, 2017: Developmental Cell
Toshiaki Izawa, Sae-Hun Park, Liang Zhao, F Ulrich Hartl, Walter Neupert
Eukaryotic cells have evolved extensive protein quality-control mechanisms to remove faulty translation products. Here, we show that yeast cells continually produce faulty mitochondrial polypeptides that stall on the ribosome during translation but are imported into the mitochondria. The cytosolic protein Vms1, together with the E3 ligase Ltn1, protects against the mitochondrial toxicity of these proteins and maintains cell viability under respiratory conditions. In the absence of these factors, stalled polypeptides aggregate after import and sequester critical mitochondrial chaperone and translation machinery...
November 2, 2017: Cell
Paul Huter, Stefan Arenz, Lars V Bock, Michael Graf, Jan Ole Frister, Andre Heuer, Lauri Peil, Agata L Starosta, Ingo Wohlgemuth, Frank Peske, Jiří Nováček, Otto Berninghausen, Helmut Grubmüller, Tanel Tenson, Roland Beckmann, Marina V Rodnina, Andrea C Vaiana, Daniel N Wilson
Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P...
November 2, 2017: Molecular Cell
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