Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

Abstract

We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.

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 5 a and data not shown).…”

On the origin of life in the Zinc world. 2. Validation of the hypothesis on the photosynthesizing zinc sulfide edifices as cradles of life on Earth

“…In some cases, however, only particular ions, e.g. Zn 2+ [ 156 , 157 ] or Mn 2+ [ 158 , 159 ] were found to be functional. Taking into account that Cd and Pb do not belong to the essential metals [ 128 ], the available data (see e.g.…”

Coupling of Fast and Slow Modes in the Reaction Pathway of the Minimal Hammerhead Ribozyme Cleavage

“…The conserved central bases, with few exceptions, are essential for ribozyme's catalytic activity (28). In terms of the structure (31)(32)(33)(34)(35)(36) and in terms of the cleavage chemistrykinetic studies (37)(38)(39), folding studies (29,(40)(41)(42)(43)(44), effects of ion binding and rescue (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55), elemental substitution at the cleavage site (49,56), cross-linking (28), effects of protonation and proton transfer (57)(58)(59)(60), single molecule studies (42,(61)(62)(63), phosphoryl transfer mechanism (37,(64)(65)(66)(67)(68)(69)(70), alternative reaction mechanisms (71,72...…”