A Base Change in the Catalytic Core of the Hairpin Ribozyme Perturbs Function but Not Domain Docking (original) (raw)
Related papers
Embo Journal, 1998
The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalytic structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions of the complex between open and closed forms. Results indicate that docking of the domains is required for both the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min -1 ) but not for cleavage, where docking (0.5 min -1 ) precedes a rate-limiting conformational transition or slow-reaction chemistry. Strikingly, most modifications to the RNA (such as a G ϩ1 A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preventing docking. This demonstrates directly that mutations and modifications which inhibit a step following substrate binding are not necessarily involved in catalysis. An improved kinetic description of the catalytic cycle is derived, including specific structural transitions.
Molecular dynamics in the hairpin ribozyme: Calculational and experimental aspects
2015
MOLECULAR DYNAMICS IN THE HAIRPIN RIBOZYME: CALCULATIONAL AND EXPERIMENTAL ASPECTS By Patrick Omondi Ochieng The increasing role of RNA therapy in targeting diseases has inspired several RNA studies and especially structural RNA. Of interest to many scientists is how such RNA can perform their work with limited functional groups available to RNA. The structural versatility of RNA seems to underscore the importance of dynamics in performing several functions. Ribozymes are a good example of structured RNA involved in RNA backbone cleavage with a range of strategies. Hairpin ribozyme invokes domain-domain docking to activate the cleavage process. The major loop rearrangements observed upon docking, as well as the kinetically unfavorable docking process, both argue for conformational selection by pre-organization of the catalytically-competent active site of the hairpin ribozyme. In this thesis, we sought to study the behavior of loop A in sampling the docked-like conformation as evide...
In the fluorescent spotlight: Global and local conformational changes of small catalytic RNAs
Biopolymers, 2002
RNA is a ubiquitous biopolymer that performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. RNA is unique in that it can carry both genetic information and catalytic function. Its secondary structure domains, which fold stably and independently, assemble hierarchically into modular tertiary structures. Studies of these folding events are key to understanding how catalytic RNAs (ribozymes) are able to position reaction components for site-specific chemistry. We have made use of fluorescence techniques to monitor the rates and free energies of folding of the small hairpin and hepatitis delta virus (HDV) ribozymes, found in satellite RNAs of plant and the human hepatitis B viruses, respectively. In particular, fluorescence resonance energy transfer (FRET) has been employed to monitor global conformational changes, and 2-aminopurine fluorescence quenching to probe for local structural rearrangements. In this review we illuminate what we have learned about the reaction pathways of the hairpin and HDV ribozymes, and how our results have complemented other biochemical and biophysical investigations. The structural transitions observed in these two small catalytic RNAs are likely to be found in many other biological RNAs, and the described fluorescence techniques promise to be broadly applicable.
Journal of Molecular Biology, 2001
The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.
The hairpin ribozyme substrate binding-domain: A highly constrained D-shaped conformation
Journal of Molecular Biology, 2001
The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.
Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction
Nature structural biology, 1999
The equilibrium distributions of hairpin ribozyme conformational isomers have been examined by time-resolved fluorescence resonance energy transfer. Ribozymes partition between active (docked) and inactive (extended) conformers, characterized by unique interdomain distance distributions, which define differences in folding free energy. The active tertiary structure is stabilized both by specific interactions between the catalytic and the substrate-binding domains and by the structure of the intervening helical junction. Under physiological conditions, the docking equilibrium of the natural four-way junction dramatically favors the active conformer, while those of a three-way and the two-way junction used in gene therapy applications favor the inactive conformer.
The hairpin ribozyme substrate binding-domain: A highly constrained D-shaped conformation1
Journal of Molecular Biology, 2001
The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.
Journal of the American Chemical Society, 2014
The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Residues A38 and G8 have been implicated as playing roles in general acid and base catalysis, respectively. Here we explore the structure and dynamics of key active site residues using more than 1 μs of molecular dynamics simulations of the hairpin ribozyme at different stages along the catalytic pathway. Analysis of results indicates hydrogen bond interactions between the nucleophile and proR nonbridging oxygen are correlated with active inline attack conformations. Further, the simulation results suggest a possible alternative role for G8 to promote inline fitness and facilitate activation of the nucleophile by hydrogen bonding, although this does not necessarily exclude an additional role as a general base. Finally, we suggest that substitution of G8 with N7- or N3-deazaguanosine which have elevated pKa values, both with and without thio modifications at the 5&#...
Articles The Solvent-Protected Core of the Hairpin Ribozyme-Substrate Complex†
1998
ABSTRACT: The complex between the hairpin ribozyme and its substrate consists of two domains that must interact in order to form a catalytic complex, yet experimental evidence concerning the points of interaction between the two domains has been lacking. Here, we report the use of hydroxyl radical footprinting to define the interface between the two domains. Cations that support very efficient ribozyme catalysis (magnesium and cobalt(III) hexammine) lead to the formation of a docked complex that features several regions of protection, indicating a solvent-inaccessible core within the tertiary structure of the complex. Cations that are suboptimal in cleavage reactions do not produce complexes with regions of reduced solvent accessibility. Nucleotides encompassing the substrate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected, suggesting their internalization into the catalytic core. Four distinct segments of the ribozyme are protected, including G11-A14, C25-C27, A38, an...
Biochemistry, 2009
The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k obs for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the imino moiety of A9 promotes a key water-mediated contact that favors transition-state formation, which suggests an enhanced chemical repertoire for RNA.