![]() ![]() AcrIE4-F7 is encoded by a mobile genetic element in Pseudomonas citronellolis. However, it is homologous to the N-terminal domain (NTD) of a fusion Acr protein, AcrIE4-F7, of which we have previously investigated the structure and function. To our knowledge, AcrIE4 has not been characterized biochemically. Nevertheless, in their transcriptional reporter assays, the presence of AcrIE2 did not prevent DNA binding of Cascade, suggesting a unique inhibition strategy of AcrIE2. demonstrated that AcrIE2 interacts with Cascade and not with Cas3. Co-purification experiments by Mejdani et al. The structures of AcrIE2 have been determined previously using NMR spectroscopy and X-ray crystallography with ab initio phasing. ![]() AcrIE2 and AcrIE4 were among those discovered in Pseudomonas phages. Several Acr proteins that act against type I-E CRISPR–Cas systems have been found. To counteract CRISPR-mediated immunity, bacteriophages encode anti-CRISPR (Acr) proteins that neutralize the host CRISPR–Cas systems. In type I-E CRISPR–Cas systems, which is one of the most extensively studied subtypes, five Cas proteins (Cas5e, Cas6e, Cas7e, Cas8e, and Cas11) associate with crRNAs to form a CRISPR-associated complex for antiviral defense (Cascade) that recognizes target DNA sequences and directs their degradation by Cas3 nucleases. The type I CRISPR–Cas systems are broadly distributed in various prokaryotic genomes and can be divided into several subtypes depending on their signature Cas components. In class 1 (types I, III and IV) systems, multiple Cas proteins participate in the formation of the interference complex, whereas class 2 (types II, V and VI) systems use a single multi-domain Cas protein such as Cas9 or Cas12. Finally, the crRNAs assemble with Cas protein(s) to form RNA-guided interference complexes for degrading target sequences in re-invading foreign nucleic acids.ĬRISPR–Cas systems have been identified in ~ 40% of bacterial genomes and ~ 90% of archaeal genomes and can be classified into two major groups and six types. Then, the acquired DNA sequences are transcribed as a long precursor CRISPR RNA, which is further processed into mature CRISPR RNAs (crRNAs) containing a single spacer unit. First, Cas proteins form an integrase complex that cleaves and inserts the invading DNA fragments into CRISPR loci as new spacers. ĬRISPR-mediated immunity functions through three distinct stages of anti-phage defense. CRISPRs and Cas proteins constitute an RNA-based adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. Genes encoding CRISPR-associated (Cas) proteins lie adjacent to the CRISPR arrays. CRISPRs are a class of repetitive elements found in prokaryotic genomes that consist of invariable ‘repeat’ sequences interspaced with variable ‘spacer’ sequences. One such mechanism is clustered regularly interspaced short palindromic repeat (CRISPR)-mediated prokaryotic immunity against bacteriophages. ![]() Constant, intense viral infections have led to the development of numerous anti-phage defense systems in bacteria and archaea. These prokaryotic viruses are ubiquitous with their hosts in the ecosystem. Our findings corroborate and expand the knowledge on type I-E Acr proteins, illuminating diverse molecular mechanisms of inhibiting CRISPR-mediated prokaryotic anti-phage defense.īacteriophages, also known as phages, are viruses that infect bacteria and archaea. We also analyzed the interaction between AcrIE4 and its target Cas component using biochemical methods. Binding assays with type I-E Cas proteins were carried out for the target identification of AcrIE2. We determined the crystal structure of AcrIE2 using single-wavelength anomalous diffraction and performed a structural comparison with the previously reported AcrIE2 structures solved by different techniques. Here, we describe the biochemical characterization of two type I-E Acr proteins, AcrIE2 and AcrIE4. Several Acr proteins that act against type I-E CRISPR–Cas systems have been identified. In bacteria and archaea, CRISPRs and Cas proteins constitute an adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. ![]()
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