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

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Abstract
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 kcat reduced 200-fold. Systematic variation of RNA 5‘-G1C2G3C4[G5A6G7A8]G9C10G11C12-3‘ 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in kcat for A6 → dA6 but reduced kcat up to 300-fold for the other sites. Methylation of individual 2‘-hydroxyls in a similar experiment reduced kcat by as much as 3 × 10-3-fold. In stem−loop DNA, replacement of d[G5A6G7A8] with individual ribonucleotides resulted in small kinetic changes, except for the dA6 → A6 replacement for which kcat decreased 6-fold. Insertion of d[G5A6G7A8] into an RNA stem−loop or G5A6G7A8 into a DNA stem−loop reduced kcat by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem−loops in one case (dG5,dG7) decreased kcat/Km by 105-fold, while a second change (dG5,dA8) decreased kcat 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 kcat 20-fold to 1660 min-1, a value similar to RTA's kcat on intact ribosomes.