Pdf probing nonselective cation binding in the hairpin. Image showing the diversity of ribozyme structures. The small catalytic rnas include the hammerhead, the hairpin or paperclip, hepatitis delta and vs rna. Like the hammerhead ribozyme it is found in rna satellites of plant viruses. Helix 2 is fixed in length at 4 bases by the functional requirements of the hairpin ribozyme. Evidence for the role of active site residues in the hairpin.
Anchoring hairpin ribozymes to long target rnas by loop. Residues a38 and g8 have been implicated as playing roles in general acid and base catalysis, respectively. Comparison of the structures of its constituent domains free and fully assembled demonstrates that the rna undergoes extensive structural. Reactions were performed with 25 nmoll ribozyme and 100 nmoll substrate concentrations for the entire study. The structure of helix 1, loop a, and helix 2 termed stem a was solved in isolation from the rest of the ribozyme by cai and tinoco fig. Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction. Stable stemloop domains are key elements for efficiency of natural antisense rnas. Kinetic models for the hairpin ribozyme reactions, including two populations of ribozymesubstrate complexes. Comparative analysis of hairpin ribozyme structures and.
Hairpin ribozyme cleavage catalyzed by aminoglycoside. In its natural context, the hairpin ribozyme is constructed around a fourway helical junction. Cleavage and ligation studies in hairpin and hammerhead. Efficient ribozyme mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. They may catalyze selfcleavage intramolecular or incis catalysis as well as the cleavage of external substrates intermolecular or intrans catalysis ohkawa, 1995. The full text of this article is available as a pdf 105k. Quantum mechanicalmolecular mechanical simulation study of. The underlined target bases nguc are not basepaired with the ribozyme. Comparison of the structures of its constituent domains free and fully assembled demonstrates that the rna undergoes extensive structural rearrangement. Subsequently, the strands were purified using sephadex g25. Freely diffusing single hairpin ribozymes provide insights. After substrate cleavage and product dissociation, ribozyme recovers its ground state. Fluorescence resonance energy transfer fret is a very informative technique for studying the tertiary folding of the hairpin ribozyme. Here, we demonstrate that interdomain interactions are required for catalytic function by reconstitution of activity following separation of an essential, independently folding domain loop b from the substrate binding strand at a helical junction.
This was in part based on the observation that free bases restoring activity to ribozyme abasic at position 8 all possessed an exocyclic amine group. The hairpin ribozyme is found in three pathogenic, plant, satellite viruses, although the one found in the satellite virus associated with tobacco ring spot virus. However, the hairpin ribozyme was not able to cleave a majority of this substrate even when in 400fold excess data not shown. Ribozyme and substrate sequences are in uppercase and lowercase letters, respectively. Steadystate fret methods were first used to monitor the docking of loops a and b during magnesiuminduced folding n. May 20, 20 the hairpin ribozyme is an rnamotif that catalyzes rnaprocessing reactions essential forreplication of the satellite rnamolecules in which it is embedded. The catalytic activity of the hairpin ribozyme extends to both cleavage and ligation reactions. Direct measurement of the ionization state of an essential. Examples of ribozymes include the hammerhead ribozyme, the vs ribozyme, leadzyme and the hairpin ribozyme. Electrostatic interactions in the hairpin ribozyme account. We have assessed the relative contributions of uv crosslinking, sugar repuckering and alternative folding to molecular heterogeneity of the hairpin ribozyme and find that it is multifactorial and can be systematically.
It was first identified in the minus strand of the tobacco ringspot virus trsv satellite rna where it catalyzes selfcleavage and joining reactions to process the products of rolling circle virus replication into linear and circular satellite. The active conformation of the hairpin ribozyme has been. The hairpin ribozyme against the u5 site recognizes a slightly longer sequence. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long rnas. The small selfcleaving rnas, including the hairpin, hammerhead, hepatitis delta and neurospora vs ribozymes, catalyze a reversible cleavage reaction that generates 2. Two independent nmr structures have been determined for the hairpin ribozyme. A four way junction accelerates hairpin ribozyme folding via a discrete intermediate elliot tan, timothy j. Innersphere coordinated metal ions are not required, as the inert metal ion complex cobalt hexammine promotes catalysis. Burke1 1markey center for molecular genetics, department of. Hairpins recognize the sequence nnyngucnnnnnn, where n is any nucleotide and y is a pyrimidine. It has been the subject of extensive biochemical and structural studies, perhaps the most detailed for any catalytic rna to date. The hairpin ribozyme assembles from sequences within a single plant satellite rna in vivo but separate rnas can associate through intermolecular basepaired helices to form functional complexes. Resultsanddiscussion mutations in the native sequence ofthe hairpin ribozyme and substrate fig.
We have assessed the relative contributions of uv crosslinking, sugar repuckering and alternative folding to molecular heterogeneity of the hairpin ribozyme. Whereas the reaction products from family members are. Efficacy and mode of action of hammerhead and hairpin. All these catalytic rnas reversibly cleave the phosphodiester bond of substrate rna to generate 5. Crystal structure of a hairpin ribozymeinhibitor complex.
Panel a, top, secondary structure of the complex between wildtype. Jan 01, 2004 the hairpin ribozyme is a naturally occurring rna that catalyzes sequence. Pdf recent experimental work on the hairpin and hammerhead. The hairpin ribozyme is a wellstudied example of a highly structured functional rna that, despite its relatively small size of 80 nucleotides, exhibits pronounced heterogeneity. Secondary structure and sequences of the hairpin ribozyme constructs and cognate substrates. The structure of the isolated, central hairpin of the hdv. Electrochemical impedance spectroscopy is successfully utilized for label. Current status and future prospects find, read and cite all the research you need on researchgate. Evidence for the role of active site residues in the. The ribozyme catalytic core is linked to a sequence that recognizes the target gene mrna, usually an antisense probe, thus combining the two strategies fig. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic rna modification method that selectively traps substrate bound to. The rna cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. The hairpin ribozyme is a member of a family of small rna endonucleases, which includes hammerhead, human hepatitis delta virus, neurospora vs, and the leaddependent catalytic rnas. Thepercent activity values arethe cleavage rates for the mutation relative to those for the native sequence.
The substrate strands s2wj, s3wj, and s4wj were labeled radioactively with 32 p at the 5. Recent studies of the hairpin ribozyme have revealed a distinct catalytic mechanism for this small rna motif. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. Inhibition of tissue factor gene induction and activity. Mar 18, 2008 the molecular mechanism of hairpin ribozyme catalysis is studied with molecular dynamics simulations using a combined quantum mechanical and molecular mechanical qmmm potential with a recently developed semiempirical am1dphot model for phosphoryl transfer reactions. Montmorillonite protection of an uvirradiated hairpin. Hairpin ribozymes are small rna catalytic motifs naturally found in the satellite rnas of tobacco ringspot virus trsv, chicory yellow mottle virus cymov, and arabis mosaic virus armv. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis from activated monomers under very specific conditions, such as an rna polymerase ribozyme. The freeenergy landscapes were constructed by properly reweighting the replica exchange data. A mechanism is explored whereby the reaction proceeds without explicit chemical participation by conserved nucleobases in the active site. We cloned hammerhead or hairpin ribozymes into a vector that was driven by the bacteriophage t 7 or the cytomegalovirus cmv immediate early rna polymerase ii promoter. The structure of the isolated, central hairpin of the hdv antigenomic ribozyme. The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions.
The hairpin ribozyme catalyses sequencespecific cleavage of rna. The natural form of the hairpin ribozyme comprises two major structural elements. Pressure modulates the selfcleavage step of the hairpin ribozyme. The ribozyme lowers the overall free energy barrier by up to 16 kcalmol, accounting for the majority of the observed rate enhancement. Metal ions play a passive role in the hairpin ribozyme catalysed. May 30, 2007 the hairpin ribozyme is a member of a family of small rna endonucleases, which includes hammerhead, human hepatitis delta virus, neurospora vs, and the leaddependent catalytic rnas. The parameters for the adhr1 control reaction free in solution not irradiated were roughly the same of the control in the presence of clay particles, with. Characterization of hairpin ribozyme reactions springerlink. Sequences are numbered according to chowrira and burke. Viroids are free naked infectious rnas whose replication and propagation, unlike those of. Probing nonselective cation binding in the hairpin ribozyme with tbiii 1 1 edited by j. In recent years, a number of ribozymes, particularly the rather small hammerhead and hairpin ribozymes, have been designed for cleavage of therapeutically relevant targets 15.
Like the other catalytic rnas, the hairpin ribozyme reaction requires a divalent cation. Aug 05, 2003 the natural form of the hairpin ribozyme comprises two major structural elements. The active site of this natural rna results from the docking of two irregular helices. The hairpin ribozyme is a naturally occurring rna that catalyzes sequence. Pentacytidine linker joining ribozyme and substrate is indicated by broken lines. Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In contrast, the ribozyme free in solution, compared to its control reaction, performed a slower, lessefficient catalysis, with 30% selfcleavage at equilibrium and a k obs 0. The unusual ionic dependence of the hairpin ribozyme prompted us to test whether neomycin b could carry out the hairpin ribozyme cleavage reaction in the absence of metal ions. Mutagenesis of the hairpin ribozyme pamelaanderson, joseph monforte1, richard tritz, steven nesbitt, john hearst2 and arnold hampel departments of biological sciences and chemistry, northern illinois university, dekalb, il 60115, sri international, 333 ravenswood avenue, menlo park, ca94025and 2department of chemistry. Anchoring hairpin ribozymes to long target rnas by looploop. A polyribonucleotide having a thermodynamically stable loop structure and ribozyme activity and a dna that codes for the polyribonucleotide.
Efficient ribozymemediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Nucleobase catalysis in the hairpin ribozyme index of. Updated on 14september2018 references tang j, breaker rr. Reconstitution of hairpin ribozyme activity following. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. Cobalt hexammine, an exchangeinert metal complex, supports full hairpin ribozyme activity, and the ribozymes catalytic rate constants display only a shallow. In vitro selection of adeninedependent hairpin ribozymes. In this structure, the bases of loop a form a series of four noncanonical pairs. Ribozymes can be engineered to suppress the expression of genes, such as those that promote cancer or those from pathogenic viruses. Metal ion binding and the folding of the hairpin ribozyme. Structure and function of the hairpin ribozyme sciencedirect. Comparison of ab initio, dft, and semiempirical qmmm.
A, substrate binds to a homogeneous ribozyme population in two different conformations, producing active and inactive ribozymesubstrate complexes. The hairpin ribozyme is a small section of rna that can act as a ribozyme. The hairpin ribozyme was engineered so that it could connect by base pairs to the 2 sequences flanking the uguc to form helices 1 and 2. Cleavage occurs at conserved sites that first need to be identified on the target. The hairpin ribozyme is a 50nucleotide rna enzyme of unknown threedimensional structure. They are denoted as 1 the tetrahymena group i intron, 2 rnase p, 3 the hammerhead ribozyme, 4 the hairpin ribozyme, and 5 the hepatitis delta virus ribozyme. Hilbers mutational studies have shown that parts of the cisacting ribozyme are redundant and that double mutations in most.
Heterogeneous folding and function of small rna motifs. Quantum mechanicalmolecular mechanical simulation study. Ribozyme can also catalyze the formation of peptide bond between adjacent amino acid by lowering the activation entropy. Pdf on sep 1, 1996, j m burke and others published hairpin ribozyme. The molecular mechanism of hairpin ribozyme catalysis is studied with molecular dynamics simulations using a combined quantum mechanical and molecular mechanical qmmm potential with a recently developed semiempirical am1dphot model for phosphoryl transfer reactions. Whereas the reaction products from family members are similar. Here we explore the structure and dynamics of key active site residues using more than 1. Inhibition of tissue factor gene induction and activity using. A highly constrained dshaped conformation robert pinard1, dominic lambert2, joyce e. The target region must be free of secondary structure and have no proteinbinding sites. Impedimetric detection of hairpin ribozyme activity. We have analyzed the capability of stateoftheart multiscale computational approaches to provide atomicresolution electronic structure insights into possible catalytic scenarios of the hairpin ribozyme by evaluating potential and free energy surfaces of the reactions by various hybrid qmmm methods. Design of hairpin ribozyme variants with improved activity.
Ribozyme complexes can assemble in a number of ways. A fourway junction accelerates hairpin ribozyme folding. This is an advanced enzymology book and discusses most of the classical and modern aspects of enzymology in detail. In this structure, the bases of loop a form a series of four noncanonical pairs resulting in an extended helix. In the configuration that is most commonly used for enzymological studies, nonessential satellite rna sequences are simply deleted leaving ribozyme. Lilley, and taekjip ha department of physics and center for biophysics and computational biology, university of illinois at urbanachampaign, urbana, il 61801. Oct 29, 20 hairpin ribozymes are small rna catalytic motifs naturally found in the satellite rnas of tobacco ringspot virus trsv, chicory yellow mottle virus cymov, and arabis mosaic virus armv. A fourway junction accelerates hairpin ribozyme folding via. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic rna modification method that selectively traps substrate bound to the. Hairpin ribozyme design employs the same approach except that the catalytic core is larger 34 nucleotides and the ribozyme targeting domains require more specificity. A four way junction accelerates hairpin ribozyme folding. Simulations are used to derive one and twodimensional potentials of mean force to examine specific reaction paths and.
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