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

Tim K. Amukele, Vern L. Schramm

Research output: Contribution to journalArticle

18 Scopus citations

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′-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 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[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 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 k cat 20-fold to 1660 min-1, a value similar to RTA's k cat on intact ribosomes.

Original languageEnglish (US)
Pages (from-to)4913-4922
Number of pages10
JournalBiochemistry
Volume43
Issue number17
DOIs
StatePublished - May 4 2004

ASJC Scopus subject areas

  • Biochemistry

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