CRISPR-Cas III-A Csm6 CARF Domain Is a Ring Nuclease Triggering Stepwise cA Cleavage with ApA>p Formation Terminating RNase Activity.

Publication Type:

Journal Article


Mol Cell, Volume 75, Issue 5, p.944-956.e6 (2019)


<p>Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cA) from ATP and subsequent cA-mediated cleavage of single-strand RNA&nbsp;(ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that&nbsp;the Csm6 CARF domain is a ring nuclease, whereby bound cA is stepwise cleaved initially to ApApApA&gt;p and subsequently to ApA&gt;p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T.&nbsp;onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the&nbsp;cA CARF-binding pocket involved in autoinhibitory regulation of RNase activity.</p>

PDB: 6O6S (apo-Csm6), PDB: 6O6T (apo-Csm6H132A), PDB: 6O6V (cA4-Csm6 soaking), PDB: 6O6X (cA4-Csm6W14A/E337A cocrystallization), PDB: 6O6Y (cA4-Csm6 cocrystallization), PDB: 6O6Z (cA4-Csm6H381A cocrystallization), PDB: 6O70 (cA4-Csm6H132A cocrystallization), PDB: 6O71 (cdA4-Csm6 soaking), PDB: 6OV0 (A4 > p-Csm6 soaking).