Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

Abstract

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.The hammerhead ribozyme ( Figure 1A) is an RNA motif that catalyzes the cleavage of an RNA substrate via a transesterification reaction, producing a 2′,3′-cyclic phosphate and a 5′-hydroxyl terminus ( Figure 1B) (1). Divalent metal ions have been implicated directly in the hammerhead ribozyme cleavage mechanism (2-5), and the most popular mechanism has involved the abstraction of the 2′-hydroxyl proton at the cleavage site by a metal ion hydroxide (6). Divalent metal ions can also play a structural role in ribozyme activity by stabilizing the catalytically active tertiary conformation through coordination to heteroatoms, such as oxygen atoms or nitrogen atoms present in the nucleoside bases. The finding that the hammerhead ribozyme is active in the absence of divalent metal ions at high concentrations of monovalent metal ions calls into question the role of divalent metal ions in the mechanism of substrate cleavage (5,7,8).Divalent metal ion binding sites in the hammerhead ribozyme have been found by X-ray crystallography (9-13) and by NMR spectroscopy (14). One of the most extensively studied metal ion binding sites is the A9/G10.1 site which involves coordination of the metal ion to N7 of G10.1 and the pro-R P phosphate oxygen of A9 (Figure 2). The pro-R P oxygen of A9 has been implicated in hammerhead ribozyme function by replacement of the nonbridging, pro-R P oxygen at the A9 phosphate with sulfur. Substrate cleavage studies in the presence of Mg 2+ resulted in reduced † This work was supported by a grant from the National Institutes of Health (GM56947).

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

“…We have thus compared the efficiency of cleavage with different cations: Ca 2+ , Ba 2+ , Mn 2+ , Co 2+ , Zn 2+ and Pb 2+ (Figure 5 and Supplementary Figures S9, S10 and S11). Cleavage rates observed for the DIS duplex are comparable to those observed for the secondary cleavage sites in the minimal hammerhead ribozyme (47,48). In our case, the most efficient cation is Pb 2+ , which led to 42% of cleavage after 1 h at 5 mM concentration ( k pb 2 + = ∼10 −2 min −1 ), and the less efficient are Ca 2+ and Ba 2+ , which did not promote any detectable cleavage after 14 h (Figure 5a and data not shown).…”

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

“…The identification of the tandem purines, G22 and G23, as an inner-sphere metal-binding site is consistent with other metal sites observed in RNA. For example, metals binding to tandem purines are seen in crystal structures of human SRP, hammerhead ribozyme, and the leadzyme (22,29,30), suggesting that this may be a general metal binding motif in RNA. A possible model for the observed metal-binding site in P4 is presented in Fig.…”

Investigation of Mg2+- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry

“…In vitro transcription was optimized and carried out on a preparatory scale as in (23), using RNA polymerase prepared as in (24). The transcripts were purified by DPAGE as described in (25) to yield 0.36 nmol HF1 and 0.73 nmol HF16. Each purified transcript (10 μM, 50 μl) was dephosphorylated with calf intestinal alkaline phosphatase, then precipitated by addition of H 2 O (62.5 μl), NaOAc (3 M, 12.5 μl) and ethanol (375 μl, −20°C).…”