RESUMO
In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.
Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Modelos Moleculares , Bacteriófagos/metabolismo , Bacteriófagos/genética , Bacteriófagos/química , Sistemas CRISPR-Cas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/química , Biossíntese de Proteínas , Sequências Hélice-Volta-Hélice , Ribossomos/metabolismo , Ribossomos/química , Sítios de Ligação , Domínios Proteicos , Proteínas Virais/metabolismo , Proteínas Virais/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/química , RNA Viral/metabolismo , RNA Viral/genética , RNA Viral/química , Transcrição GênicaRESUMO
CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.