The hairpin ribozyme substrate binding-domain: A highly constrained D-shaped conformation (original) (raw)
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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 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.
Inter-domain cross-linking and molecular modelling of the hairpin ribozyme
Journal of Molecular Biology, 1997
The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2 H -2 H disulphide linkages of known spacing (12 or 16 A Ê ) between de®ned ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5 H -GAR-3 H -motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a``ribose zipper'', another group I ribozyme feature, formed between the hydroxyl groups of residues A 10, G 11 of domain A and C 25 , A 24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the speci®c phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.
Inter-domain cross-linking and molecular modelling of the hairpin ribozyme 1 1 Edited by A. R. Fresh
Journal of Molecular Biology, 1997
The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2 H -2 H disulphide linkages of known spacing (12 or 16 A Ê ) between de®ned ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5 H -GAR-3 H -motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a``ribose zipper'', another group I ribozyme feature, formed between the hydroxyl groups of residues A 10, G 11 of domain A and C 25 , A 24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the speci®c phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.
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&#...
A Base Change in the Catalytic Core of the Hairpin Ribozyme Perturbs Function but Not Domain Docking
Biochemistry, 2001
The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5′-GU-3′ immediately 5′ of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a common interdomain distance as measured by time-resolved fluorescence resonance energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.
Journal of Molecular Biology, 1999
The hairpin ribozyme-substrate complex contains two independently folding domains that interact with one another to form a catalytic complex. However, little is known about the key structural elements involved in these tertiary interactions. Here, we report the use of a photochemical crosslinking method to investigate the relative proximity and orientation of the two domains of the hairpin ribozyme. This method allows the incorporation of a photochemical azidophenacyl group at speci®ed positions within synthetic oligoribonucleotides. Photocrosslinking was performed following the assembly of four RNA oligonucleotides into active ribozyme-substrate complexes. Two photoagent attachment sites in the substrate binding strand within domain A (between positions A7-G8 and A10-G11) and three in the 5 H strand of domain B (A20-G21, A22-A23 and A24-C25) were studied. Several crosslinks between the substrate binding strand and the 5 H segment of domain B were detected. All of the photoagent-speci®c crosslinked species were dependent upon proper assembly and folding of the ribozyme-substrate complex. In addition, a substrate base mutation (G 1 to A 1) that prevents the docking of the two domains, blocks the crosslink formation. Four interdomain crosslinks (A7-G8/C25-A26 (two species); A10-G11/A22 and A24-C25/C12-G13) have been shown to retain catalytic activity. Taken together, these results indicate that the characterized crosslinks provide important information concerning the alignment of the two domains and accurately re¯ect the active docked conformation of the molecule.
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...
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.
Journal of Physical Chemistry B, 2010
The hairpin ribozyme is a prominent member of the group of small catalytic RNAs (RNA enzymes or ribozymes) because it does not require metal ions to achieve catalysis. Biochemical and structural data have implicated guanine 8 (G8) and adenine 38 (A38) as catalytic participants in cleavage and ligation catalyzed by the hairpin ribozyme, yet their exact role in catalysis remains disputed. To gain insight into dynamics in the active site of a minimal self-cleaving hairpin ribozyme, we have performed extensive classical, explicit-solvent molecular dynamics (MD) simulations on timescales of 50-150 ns. Starting from the available X-ray crystal structures, we investigated the structural impact of the protonation states of G8 and A38, and the inactivating A−1(2′-methoxy) substitution employed in crystallography. Our simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 profoundly distorts the active site. Thus MD simulations do not support a straightforward participation of the deprotonated G8 in catalysis. By comparison, the G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. Furthermore, a protonated A38H + is more consistent with the crystallography data than a canonical A38. The simulations thus support the notion that A38H + is the dominant form in the crystals, grown at pH 6. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the structures of active site and S-turn. Yet, we occasionally also observe formation of a stable A−1(2′-OH)…A38(N1) hydrogen bond, which documents the ability of the ribozyme to form this hydrogen bond, consistent with a potential role of A38 as general base catalyst. The presence of this hydrogen bond is, however, incompatible with the expected in-line attack angle necessary for self-cleavage, requiring a rapid transition of the deprotonated 2′-oxyanion to a position more favorable for in-line attack after proton transfer from A−1(2′-OH) to A38(N1). The simulations revealed a potential force field artifact, occasional but irreversible formation of 'ladder-like', underwound A-RNA structure in one of the external helices. Although it does not affect the catalytic center of the hairpin ribozyme, further studies are under way to better assess possible influence of such force field behavior on long RNA simulations.