Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.657
Filtrar
1.
Biochem Biophys Res Commun ; 535: 47-53, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33340765

RESUMO

The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling.


Assuntos
Ácido Benzoico/química , Peptídeos/química , Receptores de Interleucina-6/química , Ribossomos/química , Ciclização , Código Genético , Humanos , Biblioteca de Peptídeos , RNA Mensageiro , Técnica de Seleção de Aptâmeros
2.
Proc Natl Acad Sci U S A ; 117(38): 23539-23547, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907940

RESUMO

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.


Assuntos
RNA Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina
3.
Proc Natl Acad Sci U S A ; 117(33): 19879-19887, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747536

RESUMO

The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.


Assuntos
Escherichia coli/genética , RNA Bacteriano/química , RNA Ribossômico 23S/química , Ribossomos/genética , Escherichia coli/química , Escherichia coli/metabolismo , Evolução Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Ribossomos/química , Ribossomos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(36): 22157-22166, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32855298

RESUMO

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.


Assuntos
Estresse Oxidativo , Ribossomos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Mutação
5.
Mol Cell ; 79(3): 406-415.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692975

RESUMO

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.


Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
6.
Nature ; 584(7822): 640-645, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32612237

RESUMO

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.


Assuntos
Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Escherichia coli , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Hidrólise , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/ultraestrutura , Ribossomos/química , Rotação
7.
Proc Natl Acad Sci U S A ; 117(28): 16333-16338, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601241

RESUMO

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.


Assuntos
Biossíntese de Proteínas/genética , RNA de Transferência/química , RNA de Transferência/genética , Anticódon/química , Anticódon/genética , Anticódon/metabolismo , Pareamento de Bases , Códon/genética , Códon/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
8.
Nature ; 585(7824): 298-302, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32669707

RESUMO

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.


Assuntos
Nucléolo Celular/enzimologia , Nucléolo Celular/genética , DNA Ribossômico/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Ribossomos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/fisiologia , DNA Helicases/metabolismo , DNA Intergênico/genética , Humanos , Enzimas Multifuncionais/metabolismo , Biossíntese de Proteínas , Estruturas R-Loop , RNA Helicases/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Ribonuclease H/metabolismo , Ribossomos/química , Ribossomos/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia
9.
Nucleic Acids Res ; 48(15): 8663-8674, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663277

RESUMO

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.


Assuntos
Cátions Bivalentes/metabolismo , Ferro/metabolismo , Clivagem do RNA/genética , Ribossomos/genética , Sítios de Ligação , Cátions Bivalentes/química , Ferro/química , Magnésio/química , Magnésio/metabolismo , Metais/química , Metais/metabolismo , Oxirredução/efeitos dos fármacos , Ribossomos/química
10.
Nucleic Acids Res ; 48(W1): W31-W35, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32479639

RESUMO

Frequently, the complete functional units of biological molecules are assemblies of protein and nucleic acid chains. Stunning examples are the complex structures of ribosomes. Here, we present TopMatch-web, a computational tool for the study of the three-dimensional structure, function and evolution of such molecules. The unique feature of TopMatch is its ability to match the protein as well as nucleic acid chains of complete molecular assemblies simultaneously. The resulting structural alignments are visualized instantly using the high-performance molecular viewer NGL. We use the mitochondrial ribosomes of human and yeast as an example to demonstrate the capabilities of TopMatch-web. The service responds immediately, enabling the interactive study of many pairwise alignments of large molecular assemblies in a single session. TopMatch-web is freely accessible at https://topmatch.services.came.sbg.ac.at.


Assuntos
Conformação de Ácido Nucleico , Conformação Proteica , Ribossomos/química , Software , Humanos , Internet , Modelos Moleculares , Proteínas/química , RNA Ribossômico/química , Proteínas Ribossômicas/química
11.
Nucleic Acids Res ; 48(11): 5859-5872, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421779

RESUMO

Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.


Assuntos
Fracionamento Celular , Microambiente Celular , Espaço Intracelular/química , RNA/análise , RNA/metabolismo , Análise de Sequência de RNA , Transcriptoma , Animais , Extratos Celulares/química , Centrifugação com Gradiente de Concentração , Feminino , Fígado/citologia , Fígado/metabolismo , Espectrometria de Massas , Camundongos , RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Sacarose
12.
Nature ; 581(7807): 209-214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405004

RESUMO

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.


Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismo
13.
Nucleic Acids Res ; 48(11): 6367-6381, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32406923

RESUMO

By analyzing almost 120 000 dinucleotides in over 2000 nonredundant nucleic acid crystal structures, we define 96+1 diNucleotide Conformers, NtCs, which describe the geometry of RNA and DNA dinucleotides. NtC classes are grouped into 15 codes of the structural alphabet CANA (Conformational Alphabet of Nucleic Acids) to simplify symbolic annotation of the prominent structural features of NAs and their intuitive graphical display. The search for nontrivial patterns of NtCs resulted in the identification of several types of RNA loops, some of them observed for the first time. Over 30% of the nearly six million dinucleotides in the PDB cannot be assigned to any NtC class but we demonstrate that up to a half of them can be re-refined with the help of proper refinement targets. A statistical analysis of the preferences of NtCs and CANA codes for the 16 dinucleotide sequences showed that neither the NtC class AA00, which forms the scaffold of RNA structures, nor BB00, the DNA most populated class, are sequence neutral but their distributions are significantly biased. The reported automated assignment of the NtC classes and CANA codes available at dnatco.org provides a powerful tool for unbiased analysis of nucleic acid structures by structural and molecular biologists.


Assuntos
DNA/química , DNA/classificação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Nucleotídeos/química , Nucleotídeos/classificação , RNA/química , RNA/classificação , Sítios de Ligação , Biocatálise , RNA Catalítico/química , RNA Catalítico/metabolismo , Reprodutibilidade dos Testes , Ribossomos/química , Ribossomos/metabolismo , Riboswitch
14.
Nat Commun ; 11(1): 1858, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313034

RESUMO

Ribosome engineering is a powerful approach for expanding the catalytic potential of the protein synthesis apparatus. Due to the potential detriment the properties of the engineered ribosome may have on the cell, the designer ribosome needs to be functionally isolated from the translation machinery synthesizing cellular proteins. One solution to this problem was offered by Ribo-T, an engineered ribosome with tethered subunits which, while producing a desired protein, could be excluded from general translation. Here, we provide a conceptually different design of a cell with two orthogonal protein synthesis systems, where Ribo-T produces the proteome, while the dissociable ribosome is committed to the translation of a specific mRNA. The utility of this system is illustrated by generating a comprehensive collection of mutants with alterations at every rRNA nucleotide of the peptidyl transferase center and isolating gain-of-function variants that enable the ribosome to overcome the translation termination blockage imposed by an arrest peptide.


Assuntos
Bactérias/metabolismo , Engenharia de Proteínas/métodos , Ribossomos/química , Biologia Sintética/métodos , Alelos , Sistema Livre de Células , Cristalografia por Raios X , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Mutação , Peptídeos/química , Peptidil Transferases/química , Plasmídeos/genética , Biossíntese de Proteínas , Proteoma , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA Ribossômico 23S/genética , Thermus thermophilus/química
15.
Proc Natl Acad Sci U S A ; 117(19): 10271-10277, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341159

RESUMO

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.


Assuntos
Escherichia coli/metabolismo , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Viomicina/farmacologia , Antibacterianos/farmacologia , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Modelos Moleculares , Conformação Molecular , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/química
16.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 332-339, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254057

RESUMO

Confidence maps provide complementary information for interpreting cryo-EM densities as they indicate statistical significance with respect to background noise. They can be thresholded by specifying the expected false-discovery rate (FDR), and the displayed volume shows the parts of the map that have the corresponding level of significance. Here, the basic statistical concepts of confidence maps are reviewed and practical guidance is provided for their interpretation and usage inside the CCP-EM suite. Limitations of the approach are discussed and extensions towards other error criteria such as the family-wise error rate are presented. The observed map features can be rendered at a common isosurface threshold, which is particularly beneficial for the interpretation of weak and noisy densities. In the current article, a practical guide is provided to the recommended usage of confidence maps.


Assuntos
Microscopia Crioeletrônica/métodos , ATPases Bacterianas Próton-Translocadoras/química , Gráficos por Computador , Modelos Moleculares , Conformação Proteica , Ribossomos/química , Eletricidade Estática , Estatística como Assunto , Vírus do Mosaico do Tabaco , Interface Usuário-Computador
17.
Nat Protoc ; 15(5): 1707-1741, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32269381

RESUMO

Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.


Assuntos
Técnicas de Química Sintética/métodos , Anticorpos de Domínio Único/química , Bacteriófagos/química , Ribossomos/química
18.
Cell Mol Life Sci ; 77(17): 3351-3367, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32123965

RESUMO

Spinal muscular atrophy (SMA) with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease that is characterized by distal and proximal muscle weakness and diaphragmatic palsy that leads to respiratory distress. Without intervention, infants with the severe form of the disease die before 2 years of age. SMARD1 is caused by mutations in the IGHMBP2 gene that determine a deficiency in the encoded IGHMBP2 protein, which plays a critical role in motor neuron survival because of its functions in mRNA processing and maturation. Although it is rare, SMARD1 is the second most common motor neuron disease of infancy, and currently, treatment is primarily supportive. No effective therapy is available for this devastating disease, although multidisciplinary care has been an essential element of the improved quality of life and life span extension in these patients in recent years. The objectives of this review are to discuss the current understanding of SMARD1 through a summary of the presently known information regarding its clinical presentation and pathogenesis and to discuss emerging therapeutic approaches. Advances in clinical care management have significantly extended the lives of individuals affected by SMARD1 and research into the molecular mechanisms that lead to the disease has identified potential strategies for intervention that target the underlying causes of SMARD1. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to halt or possibly prevent neurodegenerative disease in SMARD1 patients. The recent approval of the first gene therapy approach for SMA associated with mutations in the SMN1 gene may be a turning point for the application of this strategy for SMARD1 and other genetic neurological diseases.


Assuntos
Proteínas de Ligação a DNA/genética , Atrofia Muscular Espinal/patologia , Síndrome do Desconforto Respiratório do Recém-Nascido/patologia , Fatores de Transcrição/genética , Animais , Terapia Baseada em Transplante de Células e Tecidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Terapia Genética , Humanos , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/transplante , Síndrome do Desconforto Respiratório do Recém-Nascido/complicações , Síndrome do Desconforto Respiratório do Recém-Nascido/genética , Síndrome do Desconforto Respiratório do Recém-Nascido/terapia , Ribossomos/química , Ribossomos/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
19.
Nat Struct Mol Biol ; 27(4): 323-332, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32203490

RESUMO

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Biossíntese de Proteínas , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/ultraestrutura , Ubiquitina-Proteína Ligases/ultraestrutura , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Peptídeos/química , Peptídeos/genética , RNA Mensageiro/genética , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Serina Endopeptidases/química , Serina Endopeptidases/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
20.
RNA ; 26(6): 715-723, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144191

RESUMO

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.


Assuntos
Antibacterianos/química , Eritromicina/análogos & derivados , Inibidores da Síntese de Proteínas/química , Ribossomos/química , Antibacterianos/farmacologia , Microscopia Crioeletrônica , Eritromicina/química , Eritromicina/farmacologia , Escherichia coli/genética , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico 23S/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA