Nucleic Acids Research, Vol 24, Issue 4 611-618, Copyright © 1996 by Oxford University Press
M Orita, F Nishikawa, T Kohno,, T Senda, Y Mitsui, E Yaeta, T Kazunari and S Nishikawa
Ricin is a cytotoxic plant protein that inactivates ribosomes by
hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA.
Recent studies showed that a four-nucleotide loop, GAGA, can function as a
minimum substrate for ricin (the first adenosine corresponds to the site of
depurination). We previously clarified the solution structure of this loop
by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678].
To elucidate further details of the structural basis for recognition of its
substrate by ricin, we studied the properties of a synthetic
dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an
RNA hairpin structure with a GdAGA loop and in which the site of
depurination is changed from adenosine to 2'-deoxyadenosine. The
N-glycosidase activity against the GdAGA loop of the A-chain of ricin was
26 times higher than that against the GAGA loop. NMR studies indicated that
the overall structure of the GdAGA loop was similar to that of the GAGA
loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it
appears that the 2'-hydroxyl group of adenosine at the depurination site
(6A) does not participate in the recognition by ricin of the substrate.
Since the 2'-hydroxyl group can potentially destabilize the developing
positive charge of the putative transition state intermediate, an
oxycarbonium ion, the electronic effect may explain, at least in part, the
faster rate of depurination of the GdAGA loop compared to that of GAGA
loop. We also show that the amino group of 7G is essential for substrate
recognition the ricin A-chain.
ARTICLES
High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein
Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan.
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