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1.
Nat Commun ; 10(1): 2925, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266960

RESUMO

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well.


Assuntos
Bacteriófagos/enzimologia , Códon de Terminação/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Regiões Promotoras Genéticas , Proteínas Virais/genética
2.
Adv Exp Med Biol ; 1140: 359-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347058

RESUMO

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca2+, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca2+ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.


Assuntos
Canais Iônicos/fisiologia , Espectrometria de Massas , Transdução de Sinais , Membrana Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteômica
3.
Nat Commun ; 10(1): 3005, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285450

RESUMO

How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.


Assuntos
Complexos Multienzimáticos/ultraestrutura , Fosfoproteínas/ultraestrutura , Proteínas Serina-Treonina Quinases/ultraestrutura , Estresse Fisiológico , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Listeria monocytogenes/fisiologia , Complexos Multienzimáticos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Domínios Proteicos/fisiologia , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Fator sigma/metabolismo , Transdução de Sinais/fisiologia
4.
Nature ; 571(7765): 429-433, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292547

RESUMO

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.


Assuntos
Chaetomium/química , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Cristalografia por Raios X , Proteínas Fúngicas/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Galactosilceramidas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica
5.
Nat Commun ; 10(1): 2519, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175275

RESUMO

The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.


Assuntos
Potássio/metabolismo , Ribossomos/ultraestrutura , Difração de Raios X , Cátions , Microscopia Crioeletrônica , Cristalização , Cristalografia por Raios X , Escherichia coli , Biossíntese de Proteínas , Conformação Proteica , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/metabolismo
6.
Nat Commun ; 10(1): 2366, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147544

RESUMO

The mature virion of the tailed bacteriophage ϕ29 is an ~33 MDa complex that contains more than 450 subunits of seven structural proteins assembling into a prolate head and a short non-contractile tail. Here, we report the near-atomic structures of the ϕ29 pre-genome packaging head (prohead), the mature virion and the genome-emptied virion. Structural comparisons suggest local rotation or oscillation of the head-tail connector upon DNA packaging and release. Termination of the DNA packaging occurs through pressure-dependent correlative positional and conformational changes in the connector. The funnel-shaped tail lower collar attaches the expanded narrow end of the connector and has a 180-Å long, 24-strand ß barrel narrow stem tube that undergoes conformational changes upon genome release. The appendages form an interlocked assembly attaching the tail around the collar. The membrane active long loops at the distal end of the tail knob exit during the late stage of infection and form the cone-shaped tip of a largely hydrophobic helix barrel, prepared for membrane penetration.


Assuntos
Fagos Bacilares/ultraestrutura , Bacillus subtilis , Vírion/ultraestrutura , Microscopia Crioeletrônica , Empacotamento do DNA
7.
Nat Commun ; 10(1): 2393, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160557

RESUMO

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. Here we report cryo-EM structures of a hyperactive ClpB variant bound to the model substrate, casein in the presence of slowly hydrolysable ATPγS, which reveal the translocation mechanism. Distinct substrate-gripping interactions are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent to the topmost NBD1 contact. NBD conformations at the seam interface reveal how ATP hydrolysis-driven substrate disengagement and re-binding are precisely tuned to drive a directional, stepwise translocation cycle.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Caseínas/metabolismo , Endopeptidase Clp/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/metabolismo , Proteínas de Choque Térmico/ultraestrutura , Transporte Proteico , Domínio AAA , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Hidrólise , Modelos Moleculares , Peptídeos/metabolismo , Agregados Proteicos , Subunidades Proteicas/metabolismo
8.
Nat Commun ; 10(1): 2386, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160591

RESUMO

The fast development of single-particle cryogenic electron microscopy (cryo-EM) has made it more feasible to obtain the 3D structure of well-behaved macromolecules with a molecular weight higher than 300 kDa at ~3 Å resolution. However, it remains a challenge to obtain the high-resolution structures of molecules smaller than 200 kDa using single-particle cryo-EM. In this work, we apply the Cs-corrector-VPP-coupled cryo-EM to study the 52 kDa streptavidin (SA) protein supported on a thin layer of graphene and embedded in vitreous ice. We are able to solve both the apo-SA and biotin-bound SA structures at near-atomic resolution using single-particle cryo-EM. We demonstrate that the method has the potential to determine the structures of molecules as small as 39 kDa.


Assuntos
Biotina/metabolismo , Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Estreptavidina/ultraestrutura , Grafite , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Conformação Molecular , Estreptavidina/metabolismo
9.
Acta Crystallogr D Struct Biol ; 75(Pt 6): 528-535, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31205015

RESUMO

Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed in a similar fashion to a synchrotron X-ray beamline. It has been open to the ESRF structural biology user community since November 2017 and will remain open during the 2019 ESRF-EBS shutdown.


Assuntos
Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/ultraestrutura , Software , Síncrotrons/instrumentação , Vírus do Mosaico do Tabaco/ultraestrutura , França , Tabaco/virologia , Raios X
10.
Nat Commun ; 10(1): 2413, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160571

RESUMO

Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca2+) influx. Here we report the cryo-electron microscopy structure of the Syt1-SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg2+)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the 'primary' interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca2+-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg2+/Ca2+) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.


Assuntos
Membrana Celular/ultraestrutura , Lipídeos de Membrana/metabolismo , Proteínas SNARE/ultraestrutura , Sinaptotagmina I/ultraestrutura , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Magnésio/metabolismo , Fusão de Membrana , Neurotransmissores/metabolismo , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sinaptotagmina I/metabolismo
11.
Nat Commun ; 10(1): 2885, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253769

RESUMO

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway.


Assuntos
Reparo do DNA , Fator de Transcrição TFIIH/metabolismo , Adenosina Trifosfatases , Animais , Linhagem Celular , Clonagem Molecular , Microscopia Crioeletrônica , DNA/química , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli , Regulação da Expressão Gênica , Humanos , Insetos , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
12.
Nat Commun ; 10(1): 2865, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253771

RESUMO

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.


Assuntos
Chaperonina com TCP-1/fisiologia , Homólogo LST8 da Proteína Associada a MTOR/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Humanos , Homólogo LST8 da Proteína Associada a MTOR/genética , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteína Regulatória Associada a mTOR/genética , Serina-Treonina Quinases TOR/genética
13.
Nat Commun ; 10(1): 2872, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253804

RESUMO

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA's two-helix finger is close to the polypeptide, while SecA's clamp interacts with the polypeptide in a sequence-independent manner by inducing a short ß-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Cristalização , Escherichia coli , Geobacillus/metabolismo , Modelos Moleculares , Conformação Proteica , Transporte Proteico , Canais de Translocação SEC/genética
14.
BMC Bioinformatics ; 20(1): 326, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31195977

RESUMO

BACKGROUND: An important task of macromolecular structure determination by cryo-electron microscopy (cryo-EM) is the identification of single particles in micrographs (particle picking). Due to the necessity of human involvement in the process, current particle picking techniques are time consuming and often result in many false positives and negatives. Adjusting the parameters to eliminate false positives often excludes true particles in certain orientations. The supervised machine learning (e.g. deep learning) methods for particle picking often need a large training dataset, which requires extensive manual annotation. Other reference-dependent methods rely on low-resolution templates for particle detection, matching and picking, and therefore, are not fully automated. These issues motivate us to develop a fully automated, unbiased framework for particle picking. RESULTS: We design a fully automated, unsupervised approach for single particle picking in cryo-EM micrographs. Our approach consists of three stages: image preprocessing, particle clustering, and particle picking. The image preprocessing is based on multiple techniques including: image averaging, normalization, cryo-EM image contrast enhancement correction (CEC), histogram equalization, restoration, adaptive histogram equalization, guided image filtering, and morphological operations. Image preprocessing significantly improves the quality of original cryo-EM images. Our particle clustering method is based on an intensity distribution model which is much faster and more accurate than traditional K-means and Fuzzy C-Means (FCM) algorithms for single particle clustering. Our particle picking method, based on image cleaning and shape detection with a modified Circular Hough Transform algorithm, effectively detects the shape and the center of each particle and creates a bounding box encapsulating the particles. CONCLUSIONS: AutoCryoPicker can automatically and effectively recognize particle-like objects from noisy cryo-EM micrographs without the need of labeled training data or human intervention making it a useful tool for cryo-EM protein structure determination.


Assuntos
Algoritmos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina não Supervisionado , Automação , Análise por Conglomerados , Software
15.
Nat Commun ; 10(1): 2856, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253774

RESUMO

Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments.


Assuntos
Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium botulinum/metabolismo , Citoesqueleto/fisiologia , Citoesqueleto de Actina , Actinas/genética , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Conformação Proteica
16.
Nat Commun ; 10(1): 2579, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189921

RESUMO

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.


Assuntos
Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Terminação Traducional da Cadeia Peptídica/fisiologia , Fatores de Terminação de Peptídeos/ultraestrutura , Domínios Proteicos/fisiologia , Sítios de Ligação/fisiologia , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Fatores de Tempo
17.
Nat Methods ; 16(7): 578, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249417
18.
Nat Commun ; 10(1): 2617, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197137

RESUMO

Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5' maturation. Here we report the cryo-EM structures of Methanocaldococcus jannaschii (Mja) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of MjaRNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3'-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by MjaRNase P. The overall organization of MjaRNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.


Assuntos
Proteínas Arqueais/ultraestrutura , Ribonuclease P/ultraestrutura , Proteínas Arqueais/metabolismo , Biocatálise , Microscopia Crioeletrônica , Holoenzimas/ultraestrutura , Methanocaldococcus/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/ultraestrutura , Ribonuclease P/metabolismo
19.
Nat Commun ; 10(1): 2653, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201319

RESUMO

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Assuntos
Sítio Alostérico/genética , Proteínas de Bactérias/genética , Ribonucleotídeo Redutases/genética , Regulação Alostérica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/ultraestrutura , Ribonucleotídeos/metabolismo , Espalhamento a Baixo Ângulo
20.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
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