Clustered regularly interspaced short palindromic repeat (CRISPR) chromosomal loci found in prokaryotes provide an adaptive immune system against bacteriophages and plasmids. CRISPR-specific endoRNases produce short RNA molecules (crRNAs) from CRISPR transcripts, which harbor sequences complementary to invasive nucleic acid elements and ensure their selective targeting by CRISPR-associated (Cas) proteins. The extreme sequence divergence of CRISPR-specific endoRNases and their RNA substrates has obscured homology-based comparison of RNA recognition and cleavage mechanisms. Here, we show that Cse3 type CRISPR-specific endoRNases bind a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognate CRISPR RNA. Cocrystal structures of Cse3-RNA complexes reveal an RNA-induced conformational change in the enzyme active site that aligns the RNA strand for site-specific cleavage. These studies provide insight into a catalytically essential RNA recognition mechanism by a large class of CRISPR-related endoRNases.

In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary "nonself" DNA sequences for destruction, while avoiding binding to "self" sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for nonself target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved three base pair motif that is required for nonself target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade.

CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed ‘naïve’ adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.

Intron removal in nuclear precursor mRNA is catalyzed through two transesterification reactions by a multi-megaDalton ribonucleoprotein machine called the spliceosome. A complex between U2 and U6 small nuclear RNAs is a core component of the spliceosome. Here we present an NMR structural analysis of a protein-free U2-U6 complex from Saccharomyces cerevisiae. The observed folding of the U2-U6 complex is a four-helix junction, in which the catalytically important AGC triad base-pairs only within U6 and not with U2. The base-pairing of the AGC triad extends the U6 intramolecular stem-loop (U6 ISL), and the NMR structure of this extended U6 ISL reveals structural similarities with domain 5 of group II self-splicing introns. The observed conformation of the four-helix junction could be relevant to the first, but not the second, step of splicing and may help to position the U6 ISL adjacent to the 5' splice site.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.