Cyclic Tetra-Adenylate (cA₄) Recognition by Csa3; Implications for an Integrated Class 1 CRISPR-Cas Immune Response...

Csa3 family transcription factors are ancillary CRISPR-associated proteins composed of N-terminal CARF domains and C-terminal winged helix-turn-helix domains. The activity of Csa3 transcription factors is thought to be controlled by cyclic oligoadenyate (cOA) second messengers produced by type III CRISPR-Cas surveillance complexes. Here we show that Saccharolobus solfataricus Csa3a recognizes cyclic tetra-adenylate (cA₄) and that Csa3a lacks self-regulating "ring nuclease" activity present in some other CARF domain proteins. The crystal structure of the Csa3a/cA4 complex was also determined and the structural and thermodynamic basis for cA₄ recognition are described, as are conformational changes in Csa3a associated with cA₄ binding. We also characterized the effect of cA₄ on recognition of putative DNA binding sites. Csa3a binds to putative promoter sequences in a nonspecific, cooperative and cA₄-independent manner, suggesting a more complex mode of transcriptional regulation. We conclude the Csa3a/cA₄ interaction represents a nexus between the type I and type III CRISPR-Cas systems present in S. solfataricus, and discuss the role of the Csa3/cA₄ interaction in coordinating different arms of this integrated class 1 immune system to mount a synergistic, highly orchestrated immune response.