Abstract
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.
Highlights
Csa3a and cA4 stocks were centrifuged at 17,000× g for 10 min to remove any aggregates, and the UV absorbance of the samples was measured to determine concentrations (ε280nm for Csa3a = 8960 M−1 cm−1 and ε259nm for cA4 = 54,000 M−1 cm−1 ), with samples prepared by dilution into the same buffer (25 μm Csa3a dimer, 250 μM or 500 μM cA4 )
We began our investigation with further analysis of CRISPR-Cas systems in S. solfataricus
When we analyzed these sequences with MEME, we identified a consensus 18-bp palindromic motif that is conserved in 27 members of the order Sulfolobales (Figure 1A). This motif is generally absent from the promoters of non-CRISPR-associated genes in S. solfataricus; i.e., it is a CRISPRassociated promoter palindrome (CAPP). To differentiate this motif from an unrelated binding site reported for Csa3b [24], we refer to this motif as CAPPa
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The basic tenets of CRISPR-Cas adaptive immunity are well established [1,2,3] This heritable, adaptive immune response is manifested in three distinct stages, (i) adaptation, (ii) crRNA maturation and (iii) interference. The CRISPR-associated proteins Cas, Cas and Cas integrate short segments of invading DNA (protospacers) into CRISPR loci. These “spacers” are separated from those of previous invaders by a short DNA repeat, giving rise to the namesake clustered regularly interspaced short palindromic repeats, or CRISPR loci. Well appreciated is the extreme diversity of CRISPR-Cas systems They are grouped into 2 distinct classes, Class 1 and Class 2, and further subdivided into 6 types and 24 different subtypes [4]. Type I-A effector complexes, known as archaeal Cascade (aCascade), target double-stranded DNA in a PAM-dependent manner [1,9], while the type III-B and III-D effector complexes target transcriptionally active DNA and mRNA in a PAM-independent manner
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