Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros

Bases de dados
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Nucleic Acids Res ; 42(12): 7884-93, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24920831

RESUMO

The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/metabolismo , DNA/química , Escherichia coli/genética
2.
Nucleic Acids Res ; 41(12): 6347-59, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23625968

RESUMO

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Movimento (Física) , Ligação Proteica , Streptococcus agalactiae
3.
RNA Biol ; 10(5): 708-15, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23392250

RESUMO

Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.


Assuntos
Proteínas Associadas a CRISPR/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Óperon , RNA Bacteriano/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Escherichia coli K12/química , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/química , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/química , Fatores de Transcrição/química
4.
J Struct Biol ; 178(3): 350-62, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22531577

RESUMO

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Streptococcus agalactiae/metabolismo , DNA/metabolismo , Ligação Proteica
5.
Mol Microbiol ; 75(6): 1495-512, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20132443

RESUMO

Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/fisiologia , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Sequências Repetidas Invertidas , Regiões Promotoras Genéticas , Sequência de Bases , DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ordem dos Genes , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , RNA Bacteriano/biossíntese , Sítio de Iniciação de Transcrição , Transcrição Gênica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA