In lots of archaea and bacteria, small RNAs produced from clustered

In lots of archaea and bacteria, small RNAs produced from clustered frequently interspaced brief palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to focus on foreign DNA for destruction. brief palindromic repeatsCCRISPR-associated (CRISPRCCas) systems are bacterial adaptive immune system systems that make use of CRISPR-derived RNAs (crRNAs) as well as Cas proteins to guard against invasive hereditary components including bacteriophages or plasmids (1C4). Within many bacterial and most archaeal genomes, CRISPR loci are transcribed as long pre-crRNAs that are processed enzymatically into 60-nt mature crRNAs (5). In association with Cas proteins, crRNAs target foreign genetic elements for destruction by base pairing to complementary sequences in phage or plasmid DNA. Ribonucleases belonging to the Cas6 clade of Repeat-Associated Mystical Proteins (RAMP), found within Type I and III CRISPRCCas systems, discuss the ability to identify and cleave a single phosphodiester bond in a short repeated sequence of the pre-crRNA transcript (1C4,6). Cas6-mediated cleavage produces mature crRNAs bearing a unique spacer-derived guide sequence flanked by repeat-derived sequences around the 5 and 3 ends (5,7,8). Cas6 enzymes are metal-independent nucleases that catalyze RNA cleavage via a mechanism including a 2C3 cyclic intermediate (8,9). Structural studies have shown that Cas6 enzymes share a common ferredoxin or RNA acknowledgement motif (RRM) fold despite having widely divergent amino acid sequences (7,8,10C12). This sequence divergence has been thought to be responsible for the ability of Cas6 enzymes to recognize different kinds of RNA substrates. Many Type I CRISPR repeat sequences have the potential to form stable hairpin structures (13), which produce the major-groove binding sites for Cas6f (PaCas6f, also known as Csy4) and Cas6e (TtCas6e, also known as Cse3 or CasE) enzymes (8,10,11,14). By contrast, a subset of Type I and Type III CRISPR systems derive their crRNAs from loci in which the repeat sequences are predicted to be unstructured. Crystallographic studies of Cas6 (PfCas6), a prototypical Cas6 enzyme that cleaves an unstructured repeat sequence, have revealed that this ribonuclease recognizes a 5 terminal region of the repeat at a considerable distance upstream of the cleavage site (15). To determine how the Cas6 enzyme family has evolved unique RNA recognition capabilities based on a conserved structural core, we investigated two Cas6 enzymes associated with CRISPR loci in which the crRNA repeat sequences are predicted to form poor hairpin structures. These enzymes, hereafter referred to as TtCas6A and TtCas6B, are each predicted to recognize a four-base pair stem-loop just upstream of the cleavage site within pre-crRNA transcripts. Five crystal structures of TtCas6A and TtCas6B, both alone and in complex with their cognate substrate and product RNAs, show that although TtCas6A and TtCas6B share nearly identical structures, they use unique modes of RNA acknowledgement. Furthermore, binding studies and kinetic assays, together with comparisons with related Cas6 crystal structures, reveal a binding mechanism in which both the stem-loop of the repeat RNA and a single-stranded upstream 5 VX-770 segment are indispensable for substrate acknowledgement, implying a functional link between two unique RNA binding surfaces in Cas6 enzymes. These findings provide an explanation for the evolutionary relationship between Cas6 enzymes with orthogonal substrate acknowledgement capabilities and suggest mechanisms by which unique substrate binding modes can evolve from a single protein scaffold. MATERIALS AND METHODS Protein expression and purification The genes encoding TtCas6A (TTHA0078) and TtCas6B (TTHB231) were amplified from genomic DNA of HB8 and cloned into customized pET-based expression vectors (pEC-K-His and TSHR pEC-K-His-MBP) using ligation-independent cloning, resulting in protein constructs in which TtCas6A or TtCas6B were fused downstream of a hexahistidine affinity tag (pEC-K-His) or a hexahistidine-maltose-binding protein (MBP) tag (pEC-K-His-MBP) and a tobacco etch computer virus protease cleavage site. R22A, R129A and H37A mutants of TtCas6A and the H23A and H42A mutants of TtCas6B were generated using the QuikChange site-directed mutagenesis method (Agilent), and point mutations VX-770 were verified by DNA sequencing. Expression plasmids were transformed into BL21 Rosetta 2 (DE3) cells (Novagen), and protein expression was induced using 200 M IPTG at an optical cell density (OD600) of 0.7, followed by shaking at 18C for 16 h. Cells were harvested and lysed by sonication in 20 mM Tris-HCl (pH 8.0), 250 mM KCl, 20 mM imidazole, supplemented with 0.2 mg/ml lysozyme and protease inhibitors (Roche). For cleavage assays and crystallographic purposes, the proteins were purified as N-terminal hexahistidine VX-770 fusions as follows. The cleared lysate was incubated with Ni-NTA affinity resin (Qiagen) in 20 VX-770 mM Tris-HCl (pH 8.0), 250 mM KCl and 20 mM imidazole, and hexahistidine-tagged protein was eluted with 250 mM imidazole. Eluted proteins were then dialyzed against 20 mM Tris-HCl (pH.