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1.
Nat Commun ; 11(1): 4941, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009412

RESUMO

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.


Assuntos
Terminação Traducional da Cadeia Peptídica , Preparações Farmacêuticas/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Mutação/genética , Conformação Proteica , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
2.
Nat Commun ; 11(1): 5001, 2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-33020480

RESUMO

To perform their computational function, genetic circuits change states through a symphony of genetic parts that turn regulator expression on and off. Debugging is frustrated by an inability to characterize parts in the context of the circuit and identify the origins of failures. Here, we take snapshots of a large genetic circuit in different states: RNA-seq is used to visualize circuit function as a changing pattern of RNA polymerase (RNAP) flux along the DNA. Together with ribosome profiling, all 54 genetic parts (promoters, ribozymes, RBSs, terminators) are parameterized and used to inform a mathematical model that can predict circuit performance, dynamics, and robustness. The circuit behaves as designed; however, it is riddled with genetic errors, including cryptic sense/antisense promoters and translation, attenuation, incorrect start codons, and a failed gate. While not impacting the expected Boolean logic, they reduce the prediction accuracy and could lead to failures when the parts are used in other designs. Finally, the cellular power (RNAP and ribosome usage) required to maintain a circuit state is calculated. This work demonstrates the use of a small number of measurements to fully parameterize a regulatory circuit and quantify its impact on host.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Redes Reguladoras de Genes , Ribossomos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Teóricos , Biossíntese de Proteínas , RNA-Seq , Biologia Sintética , Transcrição Genética
3.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
4.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
5.
Nat Commun ; 11(1): 4827, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973167

RESUMO

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%-65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.


Assuntos
Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon/genética , RNA Mensageiro/química , RNA Mensageiro/fisiologia , Bactérias/classificação , Bactérias/genética , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos/genética , Iniciação Traducional da Cadeia Peptídica , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Ribossomos/metabolismo
6.
PLoS One ; 15(9): e0233197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946445

RESUMO

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.


Assuntos
Códon de Iniciação/genética , Biossíntese de Proteínas/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Composição de Bases/genética , Códon de Iniciação/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Simulação de Dinâmica Molecular , Fases de Leitura Aberta/genética , Proteína-Arginina N-Metiltransferases/biossíntese , Proteína-Arginina N-Metiltransferases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
7.
Nat Commun ; 11(1): 4304, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855412

RESUMO

Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.


Assuntos
Aminoácidos Cíclicos/metabolismo , Biossíntese Peptídica , Ribossomos/metabolismo , Aminoacilação de RNA de Transferência , Engenharia Genética , Mutação , Polimerização , RNA de Transferência/metabolismo , Ribossomos/genética
8.
PLoS One ; 15(7): e0233583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735619

RESUMO

Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.


Assuntos
Éxons/genética , Proteína Huntingtina/genética , Elongação Traducional da Cadeia Peptídica , Agregados Proteicos/genética , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Bases , Células Epiteliais , Genes Reporter , Células HEK293 , Humanos , Proteína Huntingtina/biossíntese , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Repetições Minissatélites , Peptídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
9.
Nat Commun ; 11(1): 4106, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796827

RESUMO

Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Microscopia Crioeletrônica , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribossomos/ultraestrutura
10.
Nat Commun ; 11(1): 4134, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807779

RESUMO

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Códon de Terminação/genética , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética
11.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
12.
PLoS Biol ; 18(8): e3000757, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32833957

RESUMO

In eukaryotes, conserved mechanisms ensure that cell growth is coordinated with nutrient availability. Overactive growth during nutrient limitation ("nutrient-growth dysregulation") can lead to rapid cell death. Here, we demonstrate that cells can adapt to nutrient-growth dysregulation by evolving major metabolic defects. Specifically, when yeast lysine-auxotrophic mutant lys- encountered lysine limitation, an evolutionarily novel stress, cells suffered nutrient-growth dysregulation. A subpopulation repeatedly evolved to lose the ability to synthesize organosulfurs (lys-orgS-). Organosulfurs, mainly reduced glutathione (GSH) and GSH conjugates, were released by lys- cells during lysine limitation when growth was dysregulated, but not during glucose limitation when growth was regulated. Limiting organosulfurs conferred a frequency-dependent fitness advantage to lys-orgS- by eliciting a proper slow growth program, including autophagy. Thus, nutrient-growth dysregulation is associated with rapid organosulfur release, which enables the selection of organosulfur auxotrophy to better tune cell growth to the metabolic environment. We speculate that evolutionarily novel stresses can trigger atypical release of certain metabolites, setting the stage for the evolution of new ecological interactions.


Assuntos
Adaptação Fisiológica/genética , Lisina/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Nutrientes/farmacologia , Saccharomyces cerevisiae/metabolismo , Autofagia/efeitos dos fármacos , Autofagia/genética , Evolução Biológica , Glucose/metabolismo , Glucose/farmacologia , Lisina/deficiência , Redes e Vias Metabólicas/genética , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Nutrientes/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirolimo/farmacologia , Estresse Fisiológico
13.
Nature ; 585(7823): 124-128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848247

RESUMO

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Transcrição Genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Ribossomos/metabolismo , Riboswitch/genética
14.
Science ; 369(6503): 554-557, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32732422

RESUMO

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.


Assuntos
Mycoplasma pneumoniae/metabolismo , Mycoplasma pneumoniae/ultraestrutura , Elongação Traducional da Cadeia Peptídica , Mapas de Interação de Proteínas , Transcrição Genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Bacteriano , Humanos , Mycoplasma pneumoniae/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ribossomos/metabolismo , Transcriptoma
15.
Nat Commun ; 11(1): 4258, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848127

RESUMO

Protein misfolding causes a wide spectrum of human disease, and therapies that target misfolding are transforming the clinical care of cystic fibrosis. Despite this success, however, very little is known about how disease-causing mutations affect the de novo folding landscape. Here we show that inherited, disease-causing mutations located within the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) have distinct effects on nascent polypeptides. Two of these mutations (A455E and L558S) delay compaction of the nascent NBD1 during a critical window of synthesis. The observed folding defect is highly dependent on nascent chain length as well as its attachment to the ribosome. Moreover, restoration of the NBD1 cotranslational folding defect by second site suppressor mutations also partially restores folding of full-length CFTR. These findings demonstrate that nascent folding intermediates can play an important role in disease pathogenesis and thus provide potential targets for pharmacological correction.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação , Substituição de Aminoácidos , Sítios de Ligação/genética , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Células HEK293 , Humanos , Técnicas In Vitro , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Dobramento de Proteína , Modificação Traducional de Proteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/metabolismo , Supressão Genética , Temperatura
16.
BMC Bioinformatics ; 21(1): 340, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738892

RESUMO

BACKGROUND: Ribosome profiling has been widely used for studies of translation under a large variety of cellular and physiological contexts. Many of these studies have greatly benefitted from a series of data-mining tools designed for dissection of the translatome from different aspects. However, as the studies of translation advance quickly, the current toolbox still falls in short, and more specialized tools are in urgent need for deeper and more efficient mining of the important and new features of the translation landscapes. RESULTS: Here, we present RiboMiner, a bioinformatics toolset for mining of multi-dimensional features of the translatome with ribosome profiling data. RiboMiner performs extensive quality assessment of the data and integrates a spectrum of tools for various metagene analyses of the ribosome footprints and for detailed analyses of multiple features related to translation regulation. Visualizations of all the results are available. Many of these analyses have not been provided by previous methods. RiboMiner is highly flexible, as the pipeline could be easily adapted and customized for different scopes and targets of the studies. CONCLUSIONS: Applications of RiboMiner on two published datasets did not only reproduced the main results reported before, but also generated novel insights into the translation regulation processes. Therefore, being complementary to the current tools, RiboMiner could be a valuable resource for dissections of the translation landscapes and the translation regulations by mining the ribosome profiling data more comprehensively and with higher resolution. RiboMiner is freely available at https://github.com/xryanglab/RiboMiner and https://pypi.org/project/RiboMiner .


Assuntos
Biologia Computacional/métodos , Biossíntese de Proteínas , Ribossomos/metabolismo , Software , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/genética , Códon/genética , Análise de Dados , Mineração de Dados
17.
Nat Commun ; 11(1): 4013, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782250

RESUMO

Antibiotics that interfere with translation, when combined, interact in diverse and difficult-to-predict ways. Here, we explain these interactions by "translation bottlenecks": points in the translation cycle where antibiotics block ribosomal progression. To elucidate the underlying mechanisms of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using inducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks causes these interactions. We further show that growth laws, combined with drug uptake and binding kinetics, enable the direct prediction of a large fraction of observed interactions, yet fail to predict suppression. However, varying two translation bottlenecks simultaneously supports that dense traffic of ribosomes and competition for translation factors account for the previously unexplained suppression. These results highlight the importance of "continuous epistasis" in bacterial physiology.


Assuntos
Antibacterianos/farmacologia , Modelos Teóricos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Interações Medicamentosas , Epistasia Genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
18.
Nat Commun ; 11(1): 4012, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782388

RESUMO

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Indenos/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Ribossomos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
19.
Methods Mol Biol ; 2203: 231-238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833216

RESUMO

Ribopuromycylation enables the visualization and quantitation of translation on a cellular level by immunofluorescence or in total using standard western blotting. This technique uses ribosome catalyzed puromycylation of nascent chains followed by immobilization on the ribosome by antibiotic chain elongation inhibitor emetine. Detection of puromycylated ribosome-bound nascent chains can then be achieved using a puromycin-specific antibody.


Assuntos
Coronavirus/genética , Puromicina/farmacologia , Infecções por Coronavirus , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Biossíntese de Proteínas , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo
20.
Med Sci (Paris) ; 36(8-9): 717-724, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32821048

RESUMO

Ribosome display is a powerful method for selection and molecular evolution of proteins and peptides from large libraries. Displayed proteins are recovered from target molecules in multiple rounds of selection in order to enrich specific binders with the desired properties. Nowadays, ribosome display has become one of the most widely-used display technologies thanks to its advantages over cell-display as phage display. Ribosome display is an in vitro method, in which a stable ternary complex is formed between the mRNA, the ribosome and the nascent protein. A selection cycle can be performed in a few days and bacterial transformation is not necessary. Ribosome display has been used to screen and select peptides, proteins or molecular scaffolds in order to increase their affinity, specificity, catalytic activity or stability. In this review, ribosome display systems and their applications in selection and evolution of proteins are described.


Assuntos
Sistema Livre de Células/metabolismo , Evolução Molecular Direcionada/métodos , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Sítios de Ligação/genética , Sistema Livre de Células/química , Perfilação da Expressão Gênica/tendências , Humanos , Ligação Proteica/genética , Proteínas Ribossômicas/metabolismo
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