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1.
Mol Cell ; 75(5): 996-1006.e8, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31377116

RESUMO

Cotranslational processing of newly synthesized proteins is fundamental for correct protein maturation. Protein biogenesis factors are thought to bind nascent polypeptides not before they exit the ribosomal tunnel. Here, we identify a nascent chain recognition mechanism deep inside the ribosomal tunnel by an essential eukaryotic cytosolic chaperone. The nascent polypeptide-associated complex (NAC) inserts the N-terminal tail of its ß subunit (N-ßNAC) into the ribosomal tunnel to sense substrates directly upon synthesis close to the peptidyl-transferase center. N-ßNAC escorts the growing polypeptide to the cytosol and relocates to an alternate binding site on the ribosomal surface. Using C. elegans as an in vivo model, we demonstrate that the tunnel-probing activity of NAC is essential for organismal viability and critical to regulate endoplasmic reticulum (ER) protein transport by controlling ribosome-Sec61 translocon interactions. Thus, eukaryotic protein maturation relies on the early sampling of nascent chains inside the ribosomal tunnel.


Assuntos
Proteínas de Caenorhabditis elegans/biossíntese , Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Canais de Translocação SEC/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/genética , Humanos , Ribossomos/genética , Canais de Translocação SEC/genética , Saccharomyces cerevisiae
2.
Proc Natl Acad Sci U S A ; 111(1): E44-53, 2014 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-24367111

RESUMO

In budding yeast the pachytene checkpoint 2 (Pch2) protein regulates meiotic chromosome axis structure by maintaining the domain-like organization of the synaptonemal complex proteins homolog pairing 1 (Hop1) and molecular zipper 1 (Zip1). Pch2 has also been shown to modulate meiotic double-strand break repair outcomes to favor recombination between homologs, play an important role in the progression of meiotic recombination, and maintain ribosomal DNA stability. Pch2 homologs are present in fruit flies, worms, and mammals, however the molecular mechanism of Pch2 function is unknown. In this study we provide a unique and detailed biochemical analysis of Pch2. We find that purified Pch2 is an AAA+ (ATPases associated with diverse cellular activities) protein that oligomerizes into single hexameric rings in the presence of nucleotides. In addition, we show Pch2 binds to Hop1, a critical axial component of the synaptonemal complex that establishes interhomolog repair bias, in a nucleotide-dependent fashion. Importantly, we demonstrate that Pch2 displaces Hop1 from large DNA substrates and that both ATP binding and hydrolysis by Pch2 are required for Pch2-Hop1 transactions. Based on these and previous cell biological observations, we suggest that Pch2 impacts meiotic chromosome function by directly regulating Hop1 localization.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Algoritmos , Sequência de Aminoácidos , Cromossomos/ultraestrutura , DNA/química , Teste de Complementação Genética , Glutationa Transferase/metabolismo , Hidrólise , Meiose , Dados de Sequência Molecular , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo
3.
Nucleic Acids Res ; 42(5): 3419-35, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24335279

RESUMO

Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.


Assuntos
Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , RNA de Transferência/metabolismo , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/metabolismo
4.
RNA ; 19(6): 789-802, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23611982

RESUMO

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an "early convergency model" in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , RNA Bacteriano/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Genes Bacterianos , Modelos Moleculares , Conformação de Ácido Nucleico , Fenótipo , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/genética , Especificidade da Espécie
5.
Proc Natl Acad Sci U S A ; 108(24): 9821-6, 2011 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-21628577

RESUMO

The chaplin proteins are functional amyloids found in the filamentous Streptomyces bacteria. These secreted proteins are required for the aerial development of Streptomyces coelicolor, and contribute to an intricate rodlet ultrastructure that decorates the surfaces of aerial hyphae and spores. S. coelicolor encodes eight chaplin proteins. Previous studies have revealed that only three of these proteins (ChpC, ChpE, and ChpH) are necessary for promoting aerial development, and of these three, ChpH is the primary developmental determinant. Here, we show that the model chaplin, ChpH, contains two amyloidogenic domains: one in the N terminus and one in the C terminus of the mature protein. These domains have different polymerization properties as determined using fluorescence spectroscopy, secondary structure analyses, and electron microscopy. We coupled these in vitro assays with in vivo genetic studies to probe the connection between ChpH amyloidogenesis and its biological function. Using mutational analyses, we demonstrated that both N- and C-terminal amyloid domains of ChpH were required for promoting aerial hypha formation, while the N-terminal domain was dispensable for assembly of the rodlet ultrastructure. These results suggest that there is a functional differentiation of the dual amyloid domains in the chaplin proteins.


Assuntos
Amiloide/genética , Proteínas Amiloidogênicas/genética , Proteínas de Bactérias/genética , Mutação , Streptomyces coelicolor/genética , Sequência de Aminoácidos , Amiloide/fisiologia , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas de Bactérias/ultraestrutura , Deleção de Genes , Microscopia Eletrônica , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Streptomyces coelicolor/crescimento & desenvolvimento , Streptomyces coelicolor/ultraestrutura
6.
Sci Rep ; 14(1): 1623, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238470

RESUMO

Asynchronously cycling cells pose a challenge to the accurate characterization of phase-specific gene expression. Current strategies, including RNAseq, survey the steady state gene expression across the cell cycle and are inherently limited by their inability to resolve dynamic gene regulatory networks. Single cell RNAseq (scRNAseq) can identify different cell cycle transcriptomes if enough cycling cells are present, however some cells are not amenable to scRNAseq. Therefore, we merged two powerful strategies, the CDT1 and GMNN degrons used in Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) cell cycle sensors and the ribosomal protein epitope tagging used in RiboTrap/Tag technologies to isolate cell cycle phase-specific mRNA for sequencing. The resulting cell cycle dependent, tagged ribosomal proteins (ccTaggedRP) were differentially expressed during the cell cycle, had similar subcellular locations as endogenous ribosomal proteins, incorporated into ribosomes and polysomes, and facilitated the recovery of cell cycle phase-specific RNA for sequencing. ccTaggedRP has broad applications to investigate phase-specific gene expression in complex cell populations.


Assuntos
Proteínas de Ciclo Celular , Transcriptoma , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Proteínas Ribossômicas/genética , Ribossomos/genética
7.
RNA ; 17(11): 2026-38, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21960487

RESUMO

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 3' end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/química , Escherichia coli/ultraestrutura , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Ribossômicas Menores de Bactérias/metabolismo
8.
RNA ; 17(4): 697-709, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21303937

RESUMO

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.


Assuntos
Escherichia coli/metabolismo , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Deleção de Genes , Estrutura Secundária de Proteína , RNA Ribossômico/química , RNA Ribossômico/genética , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura
9.
Nat Struct Mol Biol ; 30(6): 770-777, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37170030

RESUMO

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.


Assuntos
Retículo Endoplasmático , Glicoproteínas de Membrana , Animais , Glicoproteínas de Membrana/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Canais de Translocação SEC/metabolismo , Transporte Proteico , Mamíferos/metabolismo
10.
Nat Commun ; 14(1): 1095, 2023 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-36841832

RESUMO

Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.


Assuntos
Escherichia coli , Ribossomos , Microscopia Crioeletrônica/métodos , Escherichia coli/genética , Modelos Moleculares , Ribossomos/metabolismo , RNA Ribossômico/metabolismo , Bactérias/genética
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