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

Timothy Kien Amukele, Vern L. Schramm

Research output: Contribution to journalArticle

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
Externally publishedYes

Fingerprint

Ricin
Substrate Specificity
RNA
DNA
Cats
Substrates
Hydroxyl Radical
28S Ribosomal RNA
Castor Bean
Deoxyribonucleotides
Ribonucleotides
Methylation
Rapid thermal annealing
Adenine
Ribosomes
Scaffolds
Base Pairing
Adenosine
Catalytic Domain
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ricin A-Chain Substrate Specificity in RNA, DNA, and Hybrid Stem-Loop Structures. / Amukele, Timothy Kien; Schramm, Vern L.

In: Biochemistry®, Vol. 43, No. 17, 04.05.2004, p. 4913-4922.

Research output: Contribution to journalArticle

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