期刊
EMBO JOURNAL
卷 31, 期 12, 页码 2824-2832出版社
WILEY
DOI: 10.1038/emboj.2012.107
关键词
Cas6; CRISPR; Csy4; endoribonuclease; RNA cleavage
资金
- National Science Foundation
- National Defense Science & Engineering Graduate Research Fellowship programs
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [950971] Funding Source: National Science Foundation
CRISPR-Cas adaptive immune systems protect prokaryotes against foreign genetic elements. crRNAs derived from CRISPR loci base pair with complementary nucleic acids, leading to their destruction. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co-crystal structures of wild-type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 2'-hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate. The active site catalytic dyad lacks a general acid to protonate the leaving group and positively charged residues to stabilize the transition state, explaining why the observed catalytic rate constant is similar to 10(4)-fold slower than that of RNase A. We show that this RNA cleavage step is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition. Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product, evolutionary pressure has likely selected for substrate specificity and high-affinity crRNA interactions at the expense of rapid cleavage kinetics. The EMBO Journal (2012) 31, 2824-2832. doi:10.1038/emboj.2012.107; Published online 20 April 2012
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