Ricin A-chain activity on stem-loop and unstructured DNA substrates

Tim K. Amukele, Setu Roday, Vern L. Schramm

Research output: Contribution to journalArticle

Abstract

Ricin toxin A-chain (RTA) depurinates a single adenylate on a GAGA stem-loop region of eukaryotic 28S RNA, making it a potent toxin. Steady state rate analysis is used to establish the kinetic parameters for depurination of short RNA, DNA, and RNA-DNA hybrids of GAGA linear segments and stem-loop regions as substrates for RTA. Both stem and tetraloop structures are essential for action on RNA. For DNA stem-loop substrates, stem stability plays a small role in enhancing catalytic turnover but can enhance binding by up to 3 orders of magnitude. DNA sequences of d[GAGA] without stem-loop structures are found to be slow substrates for RTA. In contrast, equivalent RNA sequences exhibit no activity with RTA. Introduction of a deoxyadenosine at the depurination site of short RNA oligonucleotides restores catalytic function. NMR analysis indicates that the short, nonsubstrate GAGA is converted to substrate in GdAGA by the presence of a more flexible ribosyl group at the deoxyadenosine site. Conversion between C2′-endo and C2′-exo conformations at the deoxyadenosine site moves the 3'-and 5′-phosphorus atoms by 1.1 Å, and the former is proposed to place them in a catalytically favorable configuration. The ability to use short RNA-DNA hybrids as substrates for RTA permits exploration of related structures to function as substrates and inhibitors.

Original languageEnglish (US)
Pages (from-to)4416-4425
Number of pages10
JournalBiochemistry
Volume44
Issue number11
DOIs
StatePublished - Mar 22 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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