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Ricin A-Chain Activity on Stem−Loop and Unstructured DNA Substrates

2005; American Chemical Society; Volume: 44; Issue: 11 Linguagem: Inglês

10.1021/bi0474362

1943-295X

Timothy Amukele, Setu Roday, Vern L. Schramm,

Calcium signaling and nucleotide metabolism

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.