Identification and characterization of a divalent metal ion-dependent cleavage site in the hammerhead ribozyme.
We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occurs between nucleotides G8 and A9, yielding a free 5'-hydroxyl group and a 2',3'-cyclic phosphate. This cleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known to show preference for Zn(2+). Cleavage is also observed in the presence of Ni(2+), Co(2+), Mn(2+), Cd(2+), and Pb(2+), while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg(2+), Ca(2+), Sr(2+), and Ba(2+). A linear relationship between the logarithm of the rate and pH was observed for the Zn(2+)-dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to or in the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1 metal ion binding site, abstracts the proton from the 2'-hydroxyl group of G8, which attacks the A9 phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure [Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G. (1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity of the 2'-hydroxyl group to the metal ion complex.
Markley
J C
JC
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Godde
F
F
Sigurdsson
S T
ST
D000225:Adenine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002104:Cadmium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007854:Lead / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D002413:Cations, Divalent; D002414:Cations, Monovalent; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D002104:Cadmium; D007854:Lead; D003035:Cobalt; D008345:Manganese; D006147:Guanine; D009532:Nickel; D015032:Zinc; D000225:Adenine