The CRISPR endoribonuclease Csy4 utilizes unusual sequence- and structure-specific mechanisms to recognize and process crRNAs
- Author(s): Haurwitz, Rachel Elizabeth
- Advisor(s): Doudna, Jennifer A
- et al.
Many prokaryotes contain clustered regularly interspaced short palindromic repeats (CRISPRs) that together with CRISPR-associated (cas) genes confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) via enzymatic cleavage of CRISPR locus transcripts. These crRNAs serve as guides for foreign nucleic acid targeting and degradation.
Here we identify Csy4 as the endoribonuclease responsible for CRISPR transcript processing in Pseudomonas aeruginosa UCBPP-PA14. Biochemical assays and six co-crystal structures of Csy4 bound to substrate and product crRNAs reveal the complex mechanisms Csy4 utilizes to recognize, position, and cleave its cognate RNA substrate in order to generate mature crRNAs. Csy4 makes sequence-specific contacts to the major groove of its cognate RNA stem-loop and makes extensive electrostatic interactions with the phosphate backbone that are highly sensitive to the helical geometry of the substrate, resulting in an extremely high affinity binding interaction (Kd ≈ 50 pM). Csy4 has equally tight affinity for both its substrate and product RNAs and therefore functions in vivo as a single turnover catalyst. Phylogenetically conserved serine and histidine residues constitute a catalytic dyad in which the serine pins the ribosyl 2′-hydroxyl nucleophile in place, allowing the histidine to deprotonate the active site 2′-hydroxyl, leading to nucleophilic attack on the scissile phosphate. The Csy4 active site 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 ~104-fold slower than that of RNase A. The RNA cleavage step carried out by Csy4 is essential for assembly of the Csy protein-crRNA complex that facilitates target recognition. Considering that Csy4 recognizes a single cellular substrate and subsequently sequesters the cleavage product, evolutionary pressure has likely selected for substrate specificity and high-affinity crRNA interactions at the expense of rapid cleavage kinetics.
A major goal of synthetic biology is to construct reliable and predictable genetic circuits. However, synthetic genetic systems often perform unpredictably due to structural interactions between DNA, RNA, and protein components. Here we present a novel synthetic RNA processing platform utilizing Csy4 and its cognate target RNA to physically separate otherwise linked genetic elements such as promoters, ribosome binding sites, cis regulatory elements, and riboregulators. Implementation of this platform provides a general approach for creating context-free standard genetic elements that can be readily applied to the bottom-up construction of increasingly complex biological systems in a plug-and-play manner.