Ricin A-chain activity on stem-loop and unstructured DNA substrates.
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 A, 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.
Amukele
Tim K
TK
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Roday
Setu
S
Schramm
Vern L
VL
D004247:DNA / Q000138:chemical synthesis / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D003839:Deoxyadenosines; D009841:Oligonucleotides; D021122:Protein Subunits; D004247:DNA; D012276:Ricin