Trends in Biochemical Sciences
ReviewCutting it close: CRISPR-associated endoribonuclease structure and function
Section snippets
CRISPR–Cas systems and crRNA biogenesis
Most prokaryotes employ an RNA-based adaptive immune system known as CRISPR–Cas to identify and eliminate genetic parasites (reviewed in 1, 2, 3, 4, 5). Upon detecting viral or plasmid DNA in the cell, bacteria and archaea with active CRISPR systems respond by integrating short fragments of foreign DNA into the host chromosome at one end of the CRISPR locus (Figure 1) 6, 7, 8. Such loci serve as molecular vaccination cards by maintaining a genetic record of prior encounters with foreign
Core structural elements of CRISPR endoribonucleases
Cas6 family members share surprisingly limited sequence homology. Nevertheless, the many Cas6 crystal structures (Table 1) reveal a common overall fold as well as specific structural features important for crRNA binding. Cas6 enzymes consist solely of two repeat-associated mysterious protein (RAMP) domains that form ferredoxin-like or RNA recognition motif (RRM) folds, a common feature of Cas proteins 4, 9, 27, 46. Each domain has a βαββαβ secondary structure arrangement, forming a
Structural basis of RNA binding and specificity
A hallmark feature of Cas6 enzymes is the high affinity and specificity with which they bind RNA. Binding affinity appears substantially lower for Cas5c enzymes, and there are currently no available structures of Cas5c proteins bound to substrate or product RNA [22]. The structural basis of sequence- and structure-specific pre-crRNA binding by Cas6 enzymes, however, has been well characterized. The RNA-binding regions of Cas6s are positively charged, allowing the proteins to make extensive
Mechanism of cleavage and active site plasticity
In addition to highly specific RNA binding, Cas5c and Cas6 enzymes cleave RNA exclusively because their catalytic mechanism requires nucleophilic attack of the scissile phosphate by the 2′ hydroxyl group of the 5′ upstream nucleotide 14, 29, 56, 57. Structured pre-crRNAs are cleaved 3′ of the stem loop, at or near its base. Replacing the upstream nucleotide with a deoxyribonucleotide prevents cleavage but permits tight binding, making a singly deoxy-substituted pre-crRNA substrate ideal for
Interference complex assembly and crRNA loading
In the extensively studied Type I-E and I-F systems, Cas6 is an integral subunit of the targeting complex, Cascade. Recently, work on the Type I-B system showed that Cas6 is also part of the Cascade/I-B complex [34]. Cas6e and Cas6f remain tightly bound to the crRNA stem loop after cleavage, serving as a nucleation point for subsequent complex assembly 16, 29, 30, 56. In the P. aeruginosa Type I-F system, pre-crRNA processing is the requisite first step in Cascade/I-F formation 33, 57. In
Concluding remarks
Cas5c and Cas6 proteins make up a class of endoribonucleases required for the generation of mature crRNAs, which are used to target complementary nucleic acids for destruction as part of the CRISPR–Cas adaptive immune system in prokaryotes. Cas6 is the dedicated endoRNase for Type III and most Type I systems. Cas5 proteins are non-catalytic components of the Cascade interference complex in all Type I systems except the I-C subtype, in which the Cas5c variant has evolved to perform the enzymatic
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