RESUMO
Type ΙΙΙ CRISPR-Cas systems provide robust immunity against foreign RNA and DNA by sequence-specific RNase and target RNA-activated sequence-nonspecific DNase and RNase activities. We report on cryo-EM structures of Thermococcus onnurineus CsmcrRNA binary, CsmcrRNA-target RNA and CsmcrRNA-target RNAanti-tag ternary complexes in the 3.1 Å range. The topological features of the crRNA 5'-repeat tag explains the 5'-ruler mechanism for defining target cleavage sites, with accessibility of positions -2 to -5 within the 5'-repeat serving as sensors for avoidance of autoimmunity. The Csm3 thumb elements introduce periodic kinks in the crRNA-target RNA duplex, facilitating cleavage of the target RNA with 6-nt periodicity. Key Glu residues within a Csm1 loop segment of CsmcrRNA adopt a proposed autoinhibitory conformation suggestive of DNase activity regulation. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into CsmcrRNA complex assembly, mechanisms underlying RNA targeting and site-specific periodic cleavage, regulation of DNase cleavage activity, and autoimmunity suppression.
Assuntos
Autoimunidade , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/ultraestrutura , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Microscopia Crioeletrônica , Desoxirribonucleases/genética , Desoxirribonucleases/imunologia , Desoxirribonucleases/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Complexos Multiproteicos , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/imunologia , RNA Bacteriano/ultraestrutura , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/ultraestrutura , Relação Estrutura-Atividade , Thermococcus/enzimologia , Thermococcus/genética , Thermococcus/imunologiaRESUMO
Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature.
Assuntos
Proteínas Arqueais/metabolismo , Chaperoninas/metabolismo , Regulação da Expressão Gênica em Archaea , Chaperonas Moleculares/metabolismo , Thermococcus/metabolismo , Especificidade de Anticorpos/imunologia , Proteínas Arqueais/química , Proteínas Arqueais/imunologia , Western Blotting , Chaperoninas/química , Chaperoninas/imunologia , Haptenos/imunologia , Substâncias Macromoleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/imunologia , Peso Molecular , Subunidades Proteicas , Temperatura , Thermococcus/química , Thermococcus/imunologiaRESUMO
Fourteen strains of hyperthermophilic organotrophic anaerobic marine Archaea were isolated from shallow water and deep-sea hot vents, and four of them were characterized. These isolates, eight previously published strains, and six type strains of species of the order Thermococcales were selected for the study of cell wall components by means of thin sectioning or freeze-etching electron microscopy. The cell envelopes of most isolates were shown to consist of regularly arrayed surface protein layers, either single or double, with hexagonal lattice (p6) symmetry, as the exclusive constituents outside the cytoplasmic membrane. The S-layers studied differed in center-to-center spacing and molecular mass of the constituent protein subunits. Polyclonal antisera raised against the cells of 10 species were found to be species-specific and allowed 12 new isolates from shallow water hot vents to be identified as representatives of the species Thermococcus litoralis, Thermococcus stetteri, Thermococcus chitonophagus, and Thermococcus pacificus. Of the 7 deep-sea isolates, only 1 was identified as a T. litoralis strain. Thus, hyperthermophilic marine organotrophic isolates obtained from deep-sea hot vents showed greater diversity with regard to their S-layer proteins than shallow water isolates.
