Hammerheads Derived from sTRSV Show Enhanced Cleavage and Ligation Rate Constants

Nelson, Jennifer; Shepotinovskaya, Irina V.; Uhlenbeck, Olke C.

doi:10.1021/bi051130t

Cited by 52 publications

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“…All RNAs were in vitro transcribed with T7 RNA polymerase using primer-extended synthetic DNAs as templates (modified from (18)). The transcribed and co-transcriptionally cleaved portion of product was purified on denaturing polyacrylamide gels as described previously (19). The uncleaved hammerheads were transcribed at the presence of inhibitory oligonucleotides and [α- 32 P] ATP at 25° C as described previously (6,7,19,20).…”

Section: Rna Synthesismentioning

confidence: 99%

Catalytic Diversity of Extended Hammerhead Ribozymes

Shepotinovskaya

Uhlenbeck

2008

Biochemistry

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Chimeras of the well characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full length hammerhead at equilibrium and thereby deduce the elemental cleavage (k 2 ) and ligation (k -2 ) rate constants. The nine chimeras were all more active than minimal hammerheads and showed a very broad range of catalytic properties, with values of k 2 varying by 750-fold and k -2 by 100-fold. At least two of the hammerheads showed an altered dependence of k obs on magnesium concentration. Since much less catalytic diversity is observed among minimal hammerheads that lack the tertiary interactions, a possible role for the different tertiary interaction is to modulate the hammerhead cleavage properties in viroids. For example, differing hammerhead cleavage and ligation rates could affect the steady state concentrations of linear, circular, and polymeric genomes in infected cells.

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Section: Rna Synthesismentioning

confidence: 99%

Catalytic Diversity of Extended Hammerhead Ribozymes

Shepotinovskaya

Uhlenbeck

2008

Biochemistry

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“…The cleavage reaction for several natural hammerhead ribozymes has been studied (28,29,32), but the ligation kinetics have only been characterized for the sTRSV hammerhead (32).…”

Section: Author Manuscriptmentioning

confidence: 99%

“…The reaction products were separated by 20% (w/ v) denaturing PAGE. 32 P radioactivity of both ligated and unligated bands was quantified by phosphorimager analysis using a Typhoon 9400 Variable Mode Imager and ImageQuant 5.2 software (Amersham Biosciences). The observed ligation rate constants were determined with KaleidaGraph 3.5 (Synergy Software) where the fraction of 32 P-labeled ligated fulllength substrate versus time was fit to the equation F t = F o + F ∞ (1-e −kt ), where F t is the fraction of the full-length substrate at time t, F o is the initial fraction of full-length substrate, F ∞ is the maximum fraction of full-length substrate and k is the observed rate constant.…”

Section: Ligation and Cleavage Experimentsmentioning

confidence: 99%

“…In addition, Scott and coworkers have solved the X-ray structure of a construct of the Schistosoma mansoni hammerhead ribozyme, which contains the natural loop-loop tertiary interaction (40). This structure shows large conformational changes in the catalytic core compared to the structure of the minimal hammerhead ribozyme and represents an improved model for understanding the catalytic mechanism of the hammerhead ribozyme (30, 40).The cleavage reaction for several natural hammerhead ribozymes has been studied (28,29,32), but the ligation kinetics have only been characterized for the sTRSV hammerhead (32).Here we report on the ligation kinetics and internal equilibrium of the best characterized hammerhead, the Schistosoma ribozyme. This Schistosoma construct is an efficient ligase and yields up to 23% ligated product starting from the fully cleaved substrate, which is the highest level of ligation reported for any unmodified hammerhead.…”

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“…More recent studies have shown that when the minimal sequence of the hammerhead ribozyme is extended to include naturally occurring loop-loop tertiary interactions between stems I and II (Figure 1), catalysis takes place under physiological conditions both in vivo and in vitro (28,29). 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)). …”

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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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Structure–function relationships in RNA and RNP enzymes: Recent advances

Hoogstraten

Sumita

2007

Biopolymers

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The structural biology of ribozymes and ribonucleoprotein (RNP) enzymes is now sufficiently advanced that a true dialogue between structural and functional studies is possible. In this review, we consider three important systems in which an integration of structural and biochemical data has recently led to major advances in mechanistic understanding. In the hammerhead ribozyme, application‐driven biochemical studies led to the discovery of a key structural interaction that had been omitted from previously‐studied constructs. A new crystal structure of the resulting, tertiary‐stabilized hammerhead has resolved a remarkable number of longstanding paradoxes in the structure–function relationship of this ribozyme. In the Group I intron ribozyme, a flurry of high‐resolution structures has largely confirmed, but in some cases refined or challenged, a detailed model of a metalloenzyme active site that had previously been derived by meticulous quantitative metal ion rescue experiments. Finally, for the peptidyl transferase center of the ribosome, recent biochemical and chemical results motivated by the pioneering crystal structures have suggested a picture of a catalytic mechanism dominated by proximity and orientation effects and substrate‐assisted catalysis. These results refocus attention on catalysis as a property of the integrated RNP machinery as a whole, as opposed to a narrow concern with the RNA functional groups in immediate contact with the reactive center. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 317–328, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Hammerheads Derived from sTRSV Show Enhanced Cleavage and Ligation Rate Constants

Cited by 52 publications

References 38 publications

Catalytic Diversity of Extended Hammerhead Ribozymes

Catalytic Diversity of Extended Hammerhead Ribozymes

Efficient Ligation of the Schistosoma Hammerhead Ribozyme

Structure–function relationships in RNA and RNP enzymes: Recent advances

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