Ricin A-Chain Substrate Specificity in RNA, DNA, and Hybrid Stem-Loop Structures

Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A⁴³²⁴ in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k_cat reduced 200-fold. Systematic variation of RNA 5′-G₁C ₂G₃C₄[G₅A₆G ₇A₈]-G₉C₁₀G₁₁C ₁₂-3′ 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k_cat for A₆ → dA₆ but reduced k_cat up to 300-fold for the other sites. Methylation of individual 2′-hydroxyls in a similar experiment reduced k_cat by as much as 3 × 10-3-fold. In stem-loop DNA, replacement of d[G₅A₆G₇A ₈] with individual ribonucleotides resulted in small kinetic changes, except for the dA₆ → A₆ replacement for which k _cat decreased 6-fold. Insertion of d[G₅A₆G ₇A₈] into an RNA stem-loop or G₅A ₆G₇A₈ into a DNA stem-loop reduced k _cat by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG₅,dG ₇) decreased k_cat/K_m by 10⁵-fold, while a second change (dG₅,d_A8) decreased k_cat by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2′-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k _cat 20-fold to 1660 min^-1, a value similar to RTA's k _cat on intact ribosomes.