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1.
Mol Cell ; 46(2): 136-46, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22424775

RESUMO

The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ single-molecule Förster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.


Assuntos
Modelos Genéticos , RNA Polimerase II/química , Proteínas de Saccharomyces cerevisiae/química , Transcrição Gênica , Transferência Ressonante de Energia de Fluorescência , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição
2.
Genome Res ; 25(2): 155-66, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25561519

RESUMO

RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.


Assuntos
Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Mutação , RNA Polimerase III/metabolismo , Fatores Associados à Proteína de Ligação a TATA/genética , Transcrição Gênica , Anormalidades Múltiplas/diagnóstico , Adolescente , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Encéfalo/patologia , Proliferação de Células , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Imageamento por Ressonância Magnética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fenótipo , Conformação Proteica , Isoformas de Proteínas , Irmãos , Síndrome , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Peixe-Zebra
4.
J Biotechnol ; 155(3): 275-83, 2011 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-21801766

RESUMO

Protein expression in Escherichia coli is rarely trivial as low expression and insolubility are common problems. In this work we define a fusion partner, which increases expression levels similarly to the distinct function of solubility and affinity tags. This type of fusion tag we term an expressivity tag. Our work is based on earlier observations where 3' deletions of the InfB gene displays strongly increased expression levels. We have constructed progressively shortened fragments of the InfB(1-471) gene and fused gene fragments to a gfp reporter gene. A 5-fold increase in GFP expression was seen for an optimal 21 nucleotide InfB(1-21) sequence compared to gfp independently. We defined the InfB(1-21) sequence as an expressivity tag. The tag was tested for improved expression of two biotechnological important proteins streptavidin and a single chain antibody (scFv). Expression of both streptavidin and scFv(L32) was improved as evaluated by SDS-PAGE. Calculation of folding energies in the translation initiation region gave higher free energies for gfp, L32 and streptavidin when linked to InfB(1-21) than independently. InfB(1-21) did however not improve the codon usage or codon adaptation index. The expressivity tag is an important addition to the box of tools available for optimizing heterologous protein expression.


Assuntos
Biotecnologia/métodos , Clonagem Molecular/métodos , Escherichia coli/genética , Proteínas Recombinantes de Fusão/biossíntese , Animais , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Biossíntese de Proteínas , Dobramento de Proteína , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única , Solubilidade , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não Paramétricas , Termodinâmica
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