The crystal structure of an AII-RNAhammerhead ribozyme: A proposed mechanism for RNA catalytic cleavage

Abstract

We have solved the crystal structure of an all-RNA hammerhead ribozyme having a single 2'-O-methyl cytosine incorporated at the active site to prevent cleavage. The conditions used differ from those in another recent solution in four significant ways: first, it is an all-RNA ribozyme rather than a DNA-RNA hybrid; second, the connectivity of the ribozyme backbone strands is different; third, the crystals were grown in the presence of a much lower concentration of salt; and fourth, the crystal packing scheme is very different. Nevertheless, the three-dimensional structure of the all-RNA hammerhead ribozyme is similar to the previous structure. Five potential Mg(II)-binding sites are identified, including one positioned near the ribozyme catalytic pocket. Upon this basis, as well as upon comparisons with the metal-binding sites in the structurally homologous uridine turn of tRNAPhe, we propose a mechanism for RNA catalytic cleavage.

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

“…Almost all crystallographic studies to date have indicated that a metal ion (P9 metal ion), such as Mn 2 , Cd 2 or Mg 2 , is bound by the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of A 9 and N7 of G 10.1 (Pley et al 1994;Scott et al 1995). In some cases, the metal ion can be directly coordinated to N7 of G 10.1 (Scott et al 1996;Murray et al 1998a, b) and the sequence motif in such cases is known to be a metal-binding motif (Baeyens et al 1996;Peracchi et al 1998).…”

“…In particular, they can bind between the N7 of G 10.1 and the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of A 9 , modulating the activity of the ribozyme. Almost all crystallographic and biochemical studies to date have suggested that N7 of G 10.1 and the pro-Rp oxygen of the phosphate moiety of A 9 are ligated with a metal ion (the conserved P9 metal) and that this bound metal ion is catalytically important (Buzayan et al 1990;Ruffner & Uhlenbeck 1990;Ruffner et al 1990;Tuschl & Eckstein 1993;Pley et al 1994;Scott et al 1995Scott et al , 1996Peracchi et al 1996Peracchi et al , 1997Murray et al 1998a, b;Wang et al 1999;Yoshinari & Taira 2000). In some cases, another metal-binding site has been found close to G 5 and replacement here of Mg 2 by Tb 3 inhibited the cleavage reaction (Feig et al 1998).…”

“…However, in the case of the metal-binding site near G 5 , the introduction of a sterically bulky group prevented the Tb 3 ionmediated inhibition, suggesting that the Mg 2 ion at this site is bound only to the ground-state conformation and is released before the transition state is achieved (Feig et al 1998). Therefore, according to crystallographic and biochemical studies, the metal ion that is essential for catalysis appears to be the P9 metal ion [in addition to the metal ion(s) at the cleavage site], despite its considerable distance (<20 A Ê ) from the reactive phosphoryl group (Pley et al 1994;Scott et al 1995). Moreover, recent kinetic analyses demonstrated unambiguously that the added Cd 2 ion binds ®rst to the P9 position, but not with the pro-Rp phosphoryl oxygen at the cleavage site, in ribozyme-catalysed, Mg 2 -background reactions (Wang et al 1999;Yoshinari & Taira 2000).…”

Representation, searching and discovery of patterns of bases in complex RNA structures

“…The past decade has seen a vast increase in structural data on complex tertiary structures of RNA, beginning with the hammerhead ribozyme [1][2] and culminating in the atomic resolution structures of both the small (30S) [3][4] and large (50S) [5] ribosomal subunits. The recently published 5.5Å structure of the complete (70S) ribosome [6] indicates that this trend is likely to continue.…”

Influence of substrate structure on cleavage by hammerhead ribozyme

“…Comparison of sequences near the cleavage site of several self-cleaving plant RNAs has led to the identification of a consensus secondary structure termed the 'hammerhead' [6]. Recently, the three-dimensional structure of the hammerhead ha~ been described [7,8]. The hammerhead consists of three heiices of variable length and a sequence of 11 unpaired nucleotides surrounding the cleavage site.…”