Pokeweed antiviral protein (PAP) exhibits variable binding affinity to uncapped mRNA with differing structural elements

Ribosome‐inactivating proteins (RIPs) exhibit N ‐glycosidase activity by depurinating a surface exposed adenine of the α‐sarcin/ricin loop (SRL) of the large rRNA, ultimately causing cell death. They are found in numerous plant, fungi, and bacterial species and are powerful antiviral agents. PAP is a single‐chain RIP and also possesses mRNA recognition, cap binding, and depurination activities that are novel to RIPs. PAP's recognition of certain secondary structural elements of uncapped mRNA are of key interest in elucidating its enzymatic mechanism. Here, constructs of tobacco etch virus (TEV) mRNA, each containing specific structural elements, were used to study equilibrium binding. Steady state fluorescence spectroscopy was used to study binding between PAP and three distinct mRNA constructs. The K d values for the TEV mRNA constructs are given here: Full length 5′‐leader (3 pseudoknots) (FL) = 63nM, PK1 (pseudoknot 1) = 183nM and S2‐3 (a mutation in PK1) = 85nM. These results suggest that PAP recognizes specific structural elements within the mRNA for binding. The data here will be compared to equilibrium binding between PAP and FL, PK1, and S2‐3 capped TEV mRNA. This will show whether the cap structure recruits PAP at a higher affinity than the uncapped mRNA in order to place PAP within the molecular reading frame for scanning and and possibly subsequent depurination.