Characterization of a native hammerhead ribozyme derived from schistosomes

Osborne, Edith M.; Schaak, Janell E.; DeRose, Victoria J.

doi:10.1261/rna.7950605

Cited by 74 publications

(106 citation statements)

References 51 publications

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“…The sTRSV natural hammerhead also shows a higher fraction of ligated substrate than the minimal hammerhead, with K eq int ~16 in 1 mM Mg 2+ and 7.3 in 10 mM Mg 2+ (32). Previous studies on a Schistosoma construct similar to the ones used here reaches a maximum of 6% ligated substrate (with a reported K eq int of 17 at 1 mM Mg 2+ ) (39). Thus all of the natural hammerheads have lower values of K eq int than the minimal construct.…”

Section: Comparison Of the Schistosoma Hammerhead With Other Ribozymessupporting

confidence: 52%

“…This loop-loop tertiary interaction stabilizes the catalytically active conformation (30), which increases observed cleavage rate constants by up to 50-fold, allowing efficient cleavage even at low concentrations of metal ions (cleavage rate constants of ~0.2 -1.4 min −1 in 100 µM Mg 2+ (28,31,32)). The hammerhead ribozyme from the Schistosoma mansoni satellite DNA (33, 34) is the best characterized of the natural hammerhead systems with data on the cleavage kinetics, global folding by FRET, local and global dynamics by EPR, and cross-linking studies (31,(35)(36)(37)(38)(39). These studies have shown that the formation of the loop-loop tertiary interaction alters the structure, populations of active conformations, global folding and cleavage kinetics of the natural hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions.…”

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confidence: 99%

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Efficient Ligation of the Schistosoma Hammerhead Ribozyme

2007

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The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by a ~2,000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2-3 at physiological Mg 2+ ion concentrations (0.1 -1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules, and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.Ribozymes are the catalytic molecules that take part in a variety of biochemical reactions including peptidyl transfer (1, 2), self-splicing (3-5), tRNA maturation (6, 7), regulation of translation in Gram-positive bacteria (8), and processing of replication intermediates in plant pathogenic RNAs such as viroids and satellite RNAs of viruses (9-11). These plant pathogenic RNAs replicate via a rolling circle mechanism where hammerhead and hairpin ribozymes self-cleave the long multi-genomic RNA strand into single genomes (11)(12)(13)(14)(15). These linear single genomes must be subsequently re-circularized to continue the replication cycle. The hammerhead ribozyme has been proposed to also function as the RNA ligase where it reacts in cis on a linear RNA to give the circular genome (11,(16)(17)(18). Other possibilities for the in vivo ligation reaction are that the ribozyme requires the assistance of a host protein (19) or that a host protein performs the ligation reaction (20)(21)(22).The hammerhead ribozyme belongs to the group of small catalytic RNAs that includes the hairpin, VS 1 , and HDV ribozymes (23). These ribozymes catalyze the same cleavage reaction where nucleophilic attack of the 2'-OH at the cleavage site phosphate leads to products containing a 2', 3'-cyclic phosphate and a 5 24). The minimal † This work was supported in part by grants from: NIH (AI 30726) and the W. M. Keck Foundation initiative in RNA science at the University of Colorado, Boulder * To whom correspondence should be addressed. arthur.pardi@colorado.edu, Department of Chemistry and Biochemistry, 215 UCB, University of Col...

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Section: Comparison Of the Schistosoma Hammerhead With Other Ribozymessupporting

confidence: 52%

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confidence: 99%

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

2007

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“…The extended HHRz from Schistosoma mansoni demonstrates Cd 2+ rescue of P9 and scissile phosphorothioate substitutions, supporting the prediction that inner-sphere coordination of a metal ion to these phosphates is required for efficient catalysis in the stabilized HHRz (Osborne et al 2005). These prior studies were done with mixtures of phosphorothioates and fixed Cd 2+ concentrations and did not provide sufficient basis to determine ground-state affinities of the rescuing metal ions.…”

Section: Introductionmentioning

confidence: 73%

“…1) or biphasic (Eq. 2) model in which A and B represent the extents of cleavage and k obs1 and k obs2 are observed rate constants (Kim et al 2005;Osborne et al 2005). …”

Section: Hhrz Cleavage Assaysmentioning

confidence: 99%

“…Assuming that the metal FIGURE 1. (A) Secondary structure of the modified Schistosoma mansoni HHRz (MSL1L2) (Osborne et al 2005) used in these studies. (B) Crystallographic active site of the S. mansoni HHRz (2OEU) showing (1) proximity of the G12 N1, proposed to act as general base for activation of the 29 nucleophile (2, a 29-OMe in this structure) and the 29-OH of G8 that has been proposed to act as a general acid for the 59-O À leaving group (3).…”

Section: Introductionmentioning

confidence: 99%

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Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Ward¹,

DeRose²

2011

RNA

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Although the Hammerhead ribozyme (HHRz) has long been used as a model system in the field of ribozyme enzymology, several details of its mechanism are still not well understood. In particular, significant questions remain concerning the disposition and role of catalytic metals in the HHRz. Previous metal-rescue experiments using a ''minimal'' HHRz resulted in prediction of a catalytic metal that is bound in the A9/G10.1 site in the ground state of the reaction and that bridges to the scissile phosphate further along the reaction pathway. ''Native'' or extended HHRz constructs contain tertiary contacts that stabilize a more compact structure at moderate ionic strength. We performed Cd 2+ rescue experiments on an extended HHRz from Schistosoma mansoni using stereo-pure scissile phosphorothioate-substituted substrates in order to determine whether a metal ion makes contact with the scissile phosphate in the ground state or further along the reaction coordinate. Inhibition in Ca 2+ /Mg 2+ and rescue by thiophilic Cd 2+ was specific for the R p -S stereoisomer of the scissile phosphate. The affinity of the rescuing Cd 2+ , measured in two different ionic strength backgrounds, increased fourfold to 17-fold when the pro-R p oxygen is replaced by sulfur. These data support a model in which the rescuing metal ion makes a ground-state interaction with the scissile phosphate in the native HHRz. The resulting model for Mg 2+ activation in the HHRz places a metal ion in contact with the scissile phosphate, where it may provide ground-state electrostatic activation of the substrate.

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Nukleinsäuren und ihr enges Verhältnis zu Metallionen

Sigel

2007

Angewandte Chemie

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Characterization of a native hammerhead ribozyme derived from schistosomes

Cited by 74 publications

References 51 publications

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme

Nukleinsäuren und ihr enges Verhältnis zu Metallionen

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