Thank you Emma! ☺️

For more details check out our publication here: doi.org/10.1093/nar/...

The PH1 and PH2 domain loops, which span the pore of hPNPase, adopt distinct, asymmetric conformations. Only specific loop arrangements allow a loop from a neighboring protomer to interact and stabilize a single active site, suggesting an additional regulatory layer.

A catalytic magnesium ion, together with the RNA 2´-OH group, initially helps align Pi with the RNA scissile bond in position 1. Upon nucleophilic attack, the Mg²⁺ transiently shifts into position 2, avoiding steric clash with Pi and stabilizing the reaction intermediate.

For cleavage, hPNPase flips the two 3′-terminal bases of the RNA substrate by ~180°. This base flipping relocates the RNA from the loading to the pre-catalytic state, positioning the scissile bond in close proximity to Pi.

hPNPase binds RNA substrates in a largely sequence-independent manner. 3´-end specificity is enforced in the loading state that maximizes protein-RNA interactions while sterically constraining the 3´-end. To proceed to catalysis, the 3´-end must flip into the pre-catalytic state.

Our structures show that ssRNA is threaded into the active site through the bottom, and not the top pore of the trimeric hPNPase assembly.

Using cryo-EM, we determined hPNPase structures in three key states:
1. Apo state bound to inorganic phosphate (Pi)
2. RNA-bound loading state
3. RNA-bound pre-catalytic state

Using cryo-EM, we determined OBP structures at up to 2.8 Å resolution in multiple states:
1. bound to HSV-1 origin of replication (OriS) DNA

2. with a non-hydrolyzable ATP analog

3. as monomer, dimer, and higher-order assemblies