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  1 / 15972 MEDLINE  
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[PMID]:29484749
[Au] Autor:Khosravi AD; Meghdadi H; Ghadiri AA; Alami A; Sina AH; Mirsaeidi M
[Ad] Endereço:Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
[Ti] Título:rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.
[So] Source:APMIS;126(3):241-247, 2018 Mar.
[Is] ISSN:1600-0463
[Cp] País de publicação:Denmark
[La] Idioma:eng
[Ab] Resumo:The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates.
[Mh] Termos MeSH primário: Antibióticos Antituberculose/farmacologia
Proteínas de Bactérias/genética
RNA Polimerases Dirigidas por DNA/genética
Farmacorresistência Bacteriana/genética
Mycobacterium tuberculosis/genética
Rifampina/farmacologia
Tuberculose/microbiologia
[Mh] Termos MeSH secundário: Adulto
Idoso
Idoso de 80 Anos ou mais
Células Cultivadas
Feminino
Seres Humanos
Masculino
Testes de Sensibilidade Microbiana
Meia-Idade
Mycobacterium tuberculosis/efeitos dos fármacos
Mycobacterium tuberculosis/isolamento & purificação
Mutação Puntual/genética
Deleção de Sequência/genética
Adulto Jovem
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antibiotics, Antitubercular); 0 (Bacterial Proteins); 0 (rpoB protein, Mycobacterium tuberculosis); EC 2.7.7.6 (DNA-Directed RNA Polymerases); VJT6J7R4TR (Rifampin)
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180309
[Lr] Data última revisão:
180309
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180228
[St] Status:MEDLINE
[do] DOI:10.1111/apm.12804


  2 / 15972 MEDLINE  
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[PMID]:29281627
[Au] Autor:Schramm FD; Heinrich K; Thüring M; Bernhardt J; Jonas K
[Ad] Endereço:Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
[Ti] Título:An essential regulatory function of the DnaK chaperone dictates the decision between proliferation and maintenance in Caulobacter crescentus.
[So] Source:PLoS Genet;13(12):e1007148, 2017 12.
[Is] ISSN:1553-7404
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Hsp70 chaperones are well known for their important functions in maintaining protein homeostasis during thermal stress conditions. In many bacteria the Hsp70 homolog DnaK is also required for growth in the absence of stress. The molecular reasons underlying Hsp70 essentiality remain in most cases unclear. Here, we demonstrate that DnaK is essential in the α-proteobacterium Caulobacter crescentus due to its regulatory function in gene expression. Using a suppressor screen we identified mutations that allow growth in the absence of DnaK. All mutations reduced the activity of the heat shock sigma factor σ32, demonstrating that the DnaK-dependent inactivation of σ32 is a growth requirement. While most mutations occurred in the rpoH gene encoding σ32, we also identified mutations affecting σ32 activity or stability in trans, providing important new insight into the regulatory mechanisms controlling σ32 activity. Most notably, we describe a mutation in the ATP dependent protease HslUV that induces rapid degradation of σ32, and a mutation leading to increased levels of the house keeping σ70 that outcompete σ32 for binding to the RNA polymerase. We demonstrate that σ32 inhibits growth and that its unrestrained activity leads to an extensive reprogramming of global gene expression, resulting in upregulation of repair and maintenance functions and downregulation of the growth-promoting functions of protein translation, DNA replication and certain metabolic processes. While this re-allocation from proliferative to maintenance functions could provide an advantage during heat stress, it leads to growth defects under favorable conditions. We conclude that Caulobacter has co-opted the DnaK chaperone system as an essential regulator of gene expression under conditions when its folding activity is dispensable.
[Mh] Termos MeSH primário: Caulobacter crescentus/fisiologia
Proteínas de Choque Térmico HSP70/fisiologia
[Mh] Termos MeSH secundário: Proteases Dependentes de ATP/genética
Sequência de Aminoácidos
Proteínas de Bactérias/genética
Caulobacter crescentus/genética
Caulobacter crescentus/metabolismo
RNA Polimerases Dirigidas por DNA/genética
Regulação Bacteriana da Expressão Gênica
Proteínas de Choque Térmico HSP40/genética
Proteínas de Choque Térmico HSP70/genética
Proteínas de Choque Térmico HSP70/metabolismo
Resposta ao Choque Térmico
Chaperonas Moleculares/genética
Fator sigma/genética
Fatores de Transcrição/genética
Transcrição Genética/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (HSP40 Heat-Shock Proteins); 0 (HSP70 Heat-Shock Proteins); 0 (Molecular Chaperones); 0 (Sigma Factor); 0 (Transcription Factors); EC 2.7.7.6 (DNA-Directed RNA Polymerases); EC 3.4.21.- (ATP-Dependent Proteases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171228
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pgen.1007148


  3 / 15972 MEDLINE  
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[PMID]:28470600
[Au] Autor:Morra R; Young K; Casas-Mao D; Dixon N; Bird LE
[Ad] Endereço:Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.
[Ti] Título:Optimization of Membrane Protein Production Using Titratable Strains of E. coli.
[So] Source:Methods Mol Biol;1586:83-107, 2017.
[Is] ISSN:1940-6029
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The heterologous expression of membrane proteins driven by T7 RNA polymerase in E. coli is often limited by a mismatch between the transcriptional and translational rates resulting in saturation of the Sec translocon and non-insertion of the membrane protein. In order to optimize the levels of folded, functional inserted protein, it is important to correct this mismatch. In this protocol, we describe the use of titratable strains of E. coli where two small-molecule inducers are used in a bi-variate analysis to optimize the expression levels by fine tuning the transcriptional and translational rates of an eGFP-tagged membrane protein.
[Mh] Termos MeSH primário: Clonagem Molecular/métodos
Escherichia coli/genética
Proteínas de Fluorescência Verde/genética
Proteínas de Membrana/genética
[Mh] Termos MeSH secundário: Animais
RNA Polimerases Dirigidas por DNA/genética
RNA Polimerases Dirigidas por DNA/metabolismo
Escherichia coli/crescimento & desenvolvimento
Escherichia coli/metabolismo
Expressão Gênica
Proteínas de Fluorescência Verde/metabolismo
Seres Humanos
Proteínas de Membrana/metabolismo
Biossíntese de Proteínas
Proteínas Recombinantes/genética
Proteínas Recombinantes/metabolismo
Transcrição Genética
Transformação Genética
Proteínas Virais/genética
Proteínas Virais/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Membrane Proteins); 0 (Recombinant Proteins); 0 (Viral Proteins); 0 (enhanced green fluorescent protein); 147336-22-9 (Green Fluorescent Proteins); EC 2.7.7.- (bacteriophage T7 RNA polymerase); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180220
[Lr] Data última revisão:
180220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170505
[St] Status:MEDLINE
[do] DOI:10.1007/978-1-4939-6887-9_6


  4 / 15972 MEDLINE  
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[PMID]:29174734
[Au] Autor:Kenna DTD; Fuller A; Martin K; Perry C; Pike R; Burns PJ; Narayan O; Wilkinson S; Hill R; Woodford N; Logan JMJ; Turton JF
[Ad] Endereço:Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, National Infection Service, Public Health England, 61 Colindale Avenue, London NW9 5EQ, UK. Electronic address: dervla.kenna@phe.gov.uk.
[Ti] Título:rpoB gene sequencing highlights the prevalence of an E. miricola cluster over other Elizabethkingia species among UK cystic fibrosis patients.
[So] Source:Diagn Microbiol Infect Dis;90(2):109-114, 2018 Feb.
[Is] ISSN:1879-0070
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Difficulties in distinguishing species of the Elizabethkingia genus by MALDI-TOF prompted use of rpoB sequencing to investigate species distribution among 44 isolates from cystic fibrosis (CF) patients. Forty-three isolates from 38 patients formed a cluster comprising E. miricola and proposed novel species E. bruuniana sp. nov., the exception clustering with proposed species E. ursingii sp. nov., also part of this wider cluster. All 44 isolates were PCR-positive for urease gene ureG, whereas only one of 23 E. anophelis isolates from non-CF patients was positive, suggesting that this gene is largely associated with the E. miricola cluster. Antibiotic susceptibilities of 12 CF isolates revealed all were resistant to beta-lactams with the exception of piperacillin-tazobactam, and were only susceptible to minocycline and co-trimoxazole. Pulsed-field gel electrophoresis analysis revealed 4 shared strains among 17 CF patients in one pediatric clinic, but epidemiological investigations did not support patient-to-patient transmission except between one sibling pair.
[Mh] Termos MeSH primário: Proteínas de Bactérias/genética
Proteínas de Transporte/genética
Fibrose Cística/microbiologia
RNA Polimerases Dirigidas por DNA/genética
Infecções por Flavobacteriaceae/microbiologia
Flavobacteriaceae/genética
[Mh] Termos MeSH secundário: Adolescente
Criança
Pré-Escolar
Feminino
Flavobacteriaceae/classificação
Flavobacteriaceae/efeitos dos fármacos
Infecções por Flavobacteriaceae/epidemiologia
Seres Humanos
Lactente
Masculino
Testes de Sensibilidade Microbiana
Epidemiologia Molecular
Tipagem Molecular
Prevalência
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
Reino Unido/epidemiologia
Resistência beta-Lactâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Carrier Proteins); 0 (ureG protein, Bacteria); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180209
[Lr] Data última revisão:
180209
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171128
[St] Status:MEDLINE


  5 / 15972 MEDLINE  
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[PMID]:29198523
[Au] Autor:Truong DM; Boeke JD
[Ad] Endereço:Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA. Electronic address: davemtruong@gmail.com.
[Ti] Título:Resetting the Yeast Epigenome with Human Nucleosomes.
[So] Source:Cell;171(7):1508-1519.e13, 2017 Dec 14.
[Is] ISSN:1097-4172
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Humans and yeast are separated by a billion years of evolution, yet their conserved histones retain central roles in gene regulation. Here, we "reset" yeast to use core human nucleosomes in lieu of their own (a rare event taking 20 days), which initially only worked with variant H3.1. The cells adapt by acquiring suppressor mutations in cell-division genes or by acquiring certain aneuploid states. Converting five histone residues to their yeast counterparts restored robust growth. We reveal that humanized nucleosomes are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy, globally reduce RNA content, and slow adaptation to new conditions by delaying chromatin remodeling. These humanized yeasts (including H3.3) pose fundamental new questions about how chromatin is linked to many cell processes and provide a platform to study histone variants via yeast epigenome reprogramming.
[Mh] Termos MeSH primário: Histonas/química
Nucleossomos/química
Saccharomyces cerevisiae/química
[Mh] Termos MeSH secundário: Montagem e Desmontagem da Cromatina
RNA Polimerases Dirigidas por DNA/metabolismo
Regulação da Expressão Gênica
Células HeLa
Histonas/metabolismo
Seres Humanos
Mutação
Saccharomyces cerevisiae/metabolismo
Especificidade da Espécie
Transcrição Genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Histones); 0 (Nucleosomes); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1712
[Cu] Atualização por classe:171228
[Lr] Data última revisão:
171228
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171205
[St] Status:MEDLINE


  6 / 15972 MEDLINE  
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[PMID]:27779094
[Au] Autor:Böhmdorfer G; Sethuraman S; Rowley MJ; Krzyszton M; Rothi MH; Bouzit L; Wierzbicki AT
[Ad] Endereço:Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States.
[Ti] Título:Long non-coding RNA produced by RNA polymerase V determines boundaries of heterochromatin.
[So] Source:Elife;5, 2016 10 25.
[Is] ISSN:2050-084X
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:RNA-mediated transcriptional gene silencing is a conserved process where small RNAs target transposons and other sequences for repression by establishing chromatin modifications. A central element of this process are long non-coding RNAs (lncRNA), which in are produced by a specialized RNA polymerase known as Pol V. Here we show that non-coding transcription by Pol V is controlled by preexisting chromatin modifications located within the transcribed regions. Most Pol V transcripts are associated with AGO4 but are not sliced by AGO4. Pol V-dependent DNA methylation is established on both strands of DNA and is tightly restricted to Pol V-transcribed regions. This indicates that chromatin modifications are established in close proximity to Pol V. Finally, Pol V transcription is preferentially enriched on edges of silenced transposable elements, where Pol V transcribes into TEs. We propose that Pol V may play an important role in the determination of heterochromatin boundaries.
[Mh] Termos MeSH primário: Proteínas de Arabidopsis/metabolismo
Arabidopsis/enzimologia
Arabidopsis/genética
RNA Polimerases Dirigidas por DNA/metabolismo
Heterocromatina/metabolismo
RNA Longo não Codificante/metabolismo
RNA de Plantas/metabolismo
[Mh] Termos MeSH secundário: Proteínas Argonauta/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S.; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (AGO4 protein, Arabidopsis); 0 (Arabidopsis Proteins); 0 (Argonaute Proteins); 0 (Heterochromatin); 0 (RNA, Long Noncoding); 0 (RNA, Plant); EC 2.7.7.- (RNA polymerase V, Arabidopsis); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171224
[Lr] Data última revisão:
171224
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161026
[St] Status:MEDLINE


  7 / 15972 MEDLINE  
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[PMID]:29022704
[Au] Autor:Walton T; Szostak JW
[Ad] Endereço:Howard Hughes Medical Institute, Department of Molecular Biology, and Center for Computational and Integrative Biology, Massachusetts General Hospital , Boston, Massachusetts 02114, United States.
[Ti] Título:A Kinetic Model of Nonenzymatic RNA Polymerization by Cytidine-5'-phosphoro-2-aminoimidazolide.
[So] Source:Biochemistry;56(43):5739-5747, 2017 Oct 31.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The nonenzymatic polymerization of RNA may have enabled copying of functional sequences during the origin of life. Recent progress utilizing 5'-phosphoro-2-aminoimidazole activation has reinvigorated the possibility of using nonenzymatic RNA polymerization for copying arbitrary sequences. However, the reasons why 2-aminoimidazole (AI) is a superior activation group remain unclear. Here we report that the predominant mechanism of polymerization using cytidine-5'-phosphoro-2-aminoimidazolide (Cp*) involves a 2-aminoimidazolium-bridged dinucleotide (Cp*pC) intermediate. To explore the role of this intermediate, we first identify and quantify four reactions involving the synthesis and breakdown of Cp*pC that occur in the absence of the primer-template duplex. We then analyze the dependence of the rate of polymerization on the concentration of the Cp*pC intermediate in the presence and absence of the competitive inhibitor Cp. We also show that the contribution of the monomer Cp* to the polymerization rate is negligible under our primer extension conditions. Finally, we use the experimentally determined rate constants of these reactions to develop a kinetic model that helps explain the changing rate of nonenzymatic RNA polymerization over time. Our model accounts for the concentration of Cp*pC formed by Cp* under primer extension conditions. The model does not completely account for the decline in polymerization rate observed over long times, which indicates that additional important inhibitory processes have not yet been identified. Our results suggest that the superiority of 2-aminoimidazole over the traditional 2-methylimidazole activation is mostly due to the higher level of accumulation of the imidazolium-bridged intermediate under primer extension conditions.
[Mh] Termos MeSH primário: Citidina Monofosfato/análogos & derivados
Citidina Monofosfato/química
RNA Polimerases Dirigidas por DNA/química
Modelos Químicos
RNA/síntese química
[Mh] Termos MeSH secundário: Cinética
RNA/química
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
63231-63-0 (RNA); EC 2.7.7.6 (DNA-Directed RNA Polymerases); F469818O25 (Cytidine Monophosphate)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171103
[Lr] Data última revisão:
171103
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171013
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00792


  8 / 15972 MEDLINE  
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[PMID]:29020100
[Au] Autor:Barauskas O; Xing W; Aguayo E; Willkom M; Sapre A; Clarke M; Birkus G; Schultz BE; Sakowicz R; Kwon H; Feng JY
[Ad] Endereço:Gilead Sciences, Inc., Foster City, California, United States of America.
[Ti] Título:Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs.
[So] Source:PLoS One;12(10):e0185998, 2017.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.
[Mh] Termos MeSH primário: RNA Polimerases Dirigidas por DNA/metabolismo
Vírus da Influenza A/enzimologia
Nucleotídeos/metabolismo
Multimerização Proteica
Proteínas Recombinantes/metabolismo
[Mh] Termos MeSH secundário: Animais
Antivirais/farmacologia
Biocatálise/efeitos dos fármacos
Bioensaio
Replicação do DNA/efeitos dos fármacos
Replicação do DNA/genética
Cães
Eletroforese em Gel de Ágar
Vírus da Influenza A/efeitos dos fármacos
Concentração Inibidora 50
Cinética
Células Madin Darby de Rim Canino
Transcrição Genética/efeitos dos fármacos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antiviral Agents); 0 (Nucleotides); 0 (Recombinant Proteins); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171031
[Lr] Data última revisão:
171031
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171012
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0185998


  9 / 15972 MEDLINE  
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[PMID]:28991917
[Au] Autor:Schmidt ML; Hoenen T
[Ad] Endereço:Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.
[Ti] Título:Characterization of the catalytic center of the Ebola virus L polymerase.
[So] Source:PLoS Negl Trop Dis;11(10):e0005996, 2017 Oct.
[Is] ISSN:1935-2735
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:BACKGROUND: Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized. METHODOLOGY/PRINCIPAL FINDINGS: Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication. CONCLUSIONS/SIGNIFICANCE: We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle.
[Mh] Termos MeSH primário: RNA Polimerases Dirigidas por DNA/metabolismo
Ebolavirus/enzimologia
Regulação Enzimológica da Expressão Gênica/fisiologia
Regulação Viral da Expressão Gênica/fisiologia
Proteínas Virais/metabolismo
[Mh] Termos MeSH secundário: Motivos de Aminoácidos
Sequência de Aminoácidos
RNA Polimerases Dirigidas por DNA/genética
Ebolavirus/genética
Genoma Viral
Células HEK293
Seres Humanos
Proteínas Virais/genética
Replicação Viral
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Viral Proteins); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171109
[Lr] Data última revisão:
171109
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171010
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pntd.0005996


  10 / 15972 MEDLINE  
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[PMID]:28988932
[Au] Autor:Chen J; Wassarman KM; Feng S; Leon K; Feklistov A; Winkelman JT; Li Z; Walz T; Campbell EA; Darst SA
[Ad] Endereço:Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA.
[Ti] Título:6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase.
[So] Source:Mol Cell;68(2):388-397.e6, 2017 Oct 19.
[Is] ISSN:1097-4164
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ -holoenzyme (Eσ ) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.
[Mh] Termos MeSH primário: DNA de Forma B/metabolismo
DNA Bacteriano/metabolismo
RNA Polimerases Dirigidas por DNA/metabolismo
Proteínas de Escherichia coli/metabolismo
Escherichia coli/metabolismo
RNA Bacteriano/metabolismo
RNA não Traduzido/metabolismo
Fator sigma/metabolismo
[Mh] Termos MeSH secundário: DNA de Forma B/genética
DNA Bacteriano/genética
RNA Polimerases Dirigidas por DNA/genética
Escherichia coli/genética
Proteínas de Escherichia coli/genética
RNA Bacteriano/genética
RNA não Traduzido/genética
Fator sigma/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (6S RNA); 0 (DNA, B-Form); 0 (DNA, Bacterial); 0 (Escherichia coli Proteins); 0 (RNA, Bacterial); 0 (RNA, Untranslated); 0 (Sigma Factor); EC 2.7.7.- (RNA polymerase sigma 70); EC 2.7.7.6 (DNA-Directed RNA Polymerases)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:171115
[Lr] Data última revisão:
171115
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171010
[St] Status:MEDLINE



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