RESUMO
CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (â¼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.
Assuntos
Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , RNA Guia de Cinetoplastídeos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Replicação do DNA/genética , Dosagem de Genes/genéticaRESUMO
Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA Viral/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Sequência de Bases , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/metabolismo , Colífagos/química , Colífagos/genética , DNA Viral/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Dados de Sequência Molecular , Mutação , Ligação ProteicaRESUMO
CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Clivagem do RNA , Thermus thermophilus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/ultraestrutura , Endorribonucleases/química , Endorribonucleases/metabolismo , Endorribonucleases/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Thermus thermophilus/enzimologiaRESUMO
The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , RNA Bacteriano/metabolismo , Ribonucleases/metabolismo , Thermus thermophilus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/genética , Ribonucleases/química , Ribonucleases/genética , Análise de Sequência de RNA , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Thermus thermophilus/genéticaRESUMO
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation¼ - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated¼ PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
Assuntos
Sistemas CRISPR-Cas , DNA Intergênico , Escherichia coli/genéticaRESUMO
Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Viral/metabolismo , Fagos T/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Escherichia coli/virologia , Ligação Proteica , Fagos T/genéticaRESUMO
Hearing and vision sensory systems are tuned to the natural statistics of acoustic and electromagnetic energy on earth and are evolved to be sensitive in ethologically relevant ranges. But what are the natural statistics of odors, and how do olfactory systems exploit them? Dissecting an accurate machine learning model (Lee et al., 2022) for human odor perception, we find a computable representation for odor at the molecular level that can predict the odor-evoked receptor, neural, and behavioral responses of nearly all terrestrial organisms studied in olfactory neuroscience. Using this olfactory representation (principal odor map [POM]), we find that odorous compounds with similar POM representations are more likely to co-occur within a substance and be metabolically closely related; metabolic reaction sequences (Caspi et al., 2014) also follow smooth paths in POM despite large jumps in molecular structure. Just as the brain's visual representations have evolved around the natural statistics of light and shapes, the natural statistics of metabolism appear to shape the brain's representation of the olfactory world.
Assuntos
Percepção Olfatória , Receptores Odorantes , Humanos , Percepção Olfatória/fisiologia , Condutos Olfatórios/fisiologia , Olfato/fisiologia , OdorantesRESUMO
We report the whole-genome sequence of a new Escherichia coli temperate phage, Ayreon, comprising a linear double-stranded DNA (dsDNA) genome of 44,708 bp.
RESUMO
Since the discovery of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associated genes) immune systems, astonishing progress has been made on revealing their mechanistic foundations. Due to the immense potential as genome engineering tools, research has mainly focussed on a subset of Cas nucleases that target DNA. In addition, however, distinct types of RNA-targetting CRISPR-Cas systems have been identified. The focus of this review will be on the interference mechanisms of the RNA targetting type III and type VI CRISPR-Cas systems, their biological relevance and their potential for applications.
Assuntos
Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , Edição de Genes/métodos , Genoma , Imunidade Adaptativa , Animais , Bactérias/imunologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , DNA/metabolismo , Elementos de DNA Transponíveis , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Phages differ substantially in the bacterial hosts that they infect. Their host range is determined by the specific structures that they use to target bacterial cells. Tailed phages use a broad range of receptor-binding proteins, such as tail fibres, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors. Recent technical advances and new structure-function insights have begun to unravel the molecular mechanisms and temporal dynamics that govern these interactions. Here, we review the current understanding of the targeting machinery and mechanisms of tailed phages. These new insights and approaches pave the way for the application of phages in medicine and biotechnology and enable deeper understanding of their ecology and evolution.