Abstract
Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.
Highlights
Introduction sesses weak ribonuclease activity in the absence of Cyclic oligoadenylate (cOA) [10,11]
Cyclic oligoadenylate secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non- using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site
In the crenarchaea, dedicated ring nucleases degrade cA4, but the equivalent enzyme has not been identified in bacteria
Summary
Introduction sesses weak ribonuclease activity in the absence of cOA [10,11]. Once stimulated by cOA, the non-. COA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non- using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. In Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that degrades its activator.
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