Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures.
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 A(4324) 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 k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) 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 (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) 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.
Amukele
Tim K
TK
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Schramm
Vern L
VL
D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D009841:Oligonucleotides; D012339:RNA, Ribosomal, 28S; D012313:RNA; D004247:DNA; D012276:Ricin