Hammerhead ribozyme kinetics

Stage-Zimmermann, Tracy K.; Uhlenbeck, Olke C.

doi:10.1017/s1355838298980876

Cited by 193 publications

(184 citation statements)

References 97 publications

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“…Thus, the value of k obs (L) should equal k obs (C) since both reflect the sum of the same forward (k 2 ), and reverse (k -2 ) elemental rate constants (29). Furthermore, if both the cleaved and uncleaved hammerheads are fully active, the two assays should also give the same value of f eq .…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 99%

Catalytic Diversity of Extended Hammerhead Ribozymes

Shepotinovskaya

Uhlenbeck

2008

Biochemistry

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“…In this study, we use ITC (17) to determine thermodynamic parameters for the folding of the hammerhead ribozyme (18), a three-way RNA helical junction. Recently, Lilley and coworkers reported ITC-measured values for Mg 2+ -binding and coupled folding of the hammerhead ribozyme and two folding mutants (19).…”

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confidence: 99%

Entropy-Driven Folding of an RNA Helical Junction: An Isothermal Titration Calorimetric Analysis of the Hammerhead Ribozyme

2004

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Helical junctions are extremely common motifs in naturally occurring RNAs, but little is known about the thermodynamics that drive their folding. Studies of junction folding face several challenges: non-two-state folding behavior, superposition of secondary and tertiary structural energetics, and drastically opposing enthalpic and entropic contributions to folding. Here we describe a thermodynamic dissection of the folding of the hammerhead ribozyme, a three-way RNA helical junction, by using isothermal titration calorimetry of bimolecular RNA constructs. By using this method, we show that tertiary folding of the hammerhead core occurs with a highly unfavorable enthalpy change, and is therefore entropically driven. Furthermore, the enthalpies and heat capacities of core folding are the same whether supported by monovalent or divalent ions. These properties appear to be general to the core sequence of bimolecular hammerhead constructs. We present a model for the ion-induced folding of the hammerhead core that is similar to those advanced for the folding of much larger RNAs, involving ion-induced collapse to a structured, non-native state accompanied by rearrangement of core residues to produce the native fold. In agreement with previous enzymological and structural studies, our thermodynamic data suggest that the hammerhead structure is stabilized in vitro predominantly by diffusely bound ions. Our approach addresses several significant challenges that accompany the study of junction folding, and should prove useful in defining the thermodynamic determinants of stability in these important RNA motifs.RNA helical junctions, single-stranded loops flanked by multiple double helices, are pervasive in known and predicted RNA structures. Helical junctions appear in a broad range of functional RNAs (1,2), including small and large ribozymes, mRNA untranslated regions, riboregulatory RNAs, snRNAs, and rRNAs. Alone or in complex with proteins, junctions may serve as the major tertiary structural elements of small RNAs or as critical elements in organizing much larger architectures. Despite their prominence, little is known about the forces that drive the folding of RNA helical junctions. Pairwise coaxial stacking and ion-dependent folding have emerged as common themes in junction architecture, but the exact role of central, looped regions and the energetics of the folding process are still unclear. As a result, there are currently no general principles of helical junction folding dependable enough for use in predicting RNA tertiary structure, much less in predicting the impact of solution conditions or bound ligands on junction stabilities. † This work was supported by IU, the IU Department of Chemistry, grants from the NIH (GM-065430 to A.L.F. and T32-GM07757 to IU/P.J.M.), the NSF (CHE-9909407 to A.L.F.) and an HHMI/Capstone award to J.C.T. Andrew Feig is a Cottrell Scholar of Research Corporation.*To whom correspondence should be addressed:Andrew L. Feig, Department of Chemistry, Indiana University, 800 E. Kirk...

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“…This means many of the technical difficulties inherent to Laue data collection, including those mentioned above and the exacting requirement for crystals of very low mosaicity, can be avoided. For small, self-cleaving ribozymes such as the hammerhead ribozyme, typical in vitro cleavage rates of well-behaved ribozymes are on the order of one turnover per minute [4]. The turnover rate, which is highly sequence dependent, also can be modulated using pH as a controlling variable.…”

Section: Ribozymes and Crystallographic Freeze-trapping Experimentsmentioning

confidence: 99%

“…Essentially all of the enzymology experiments are interpreted in terms of a simple Michaelis-Menten mechanism in which only one intermediate, the enzyme-substrate complex, is invoked, and a simple turnover scheme in which k 2 is identified with k cat or the chemical step of the reaction is almost always assumed. The more thorough analyses [4,11] take into account enzyme-product complexes in which either or both of the product strands are associated, but these states may generally be neglected in the realm of singleturnover reactions. Because k 2 is so slow, ( 1/min), K m is identified with substrate binding and k 2 is identified with the chemical transition state in the self-cleavage reaction.…”

Section: Control Experiments Useful For Correlating Observed Structurmentioning

confidence: 99%

“…The second is that an observed pH dependence of the ratelimiting step of a ribozyme reaction entails that the ratelimiting step of the reaction is the chemical step. This assumption appears justified because proton transfer most likely occurs in the chemical step, and nucleotide bases are not normally ionizable between about pH 4 and 9 [4].…”

Section: Previous Assumptions May Be Wrongmentioning

confidence: 99%

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