Apr 12, 2001 the hairpin ribozyme catalyses sequencespecific cleavage of rna. Direct measurement of the ionization state of an essential. Crystal structure of a hairpin ribozymeinhibitor complex. 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. Catalysis by the hairpin ribozyme is stimulated by a wide range of both simple and complex metallic and organic cations. This independence from divalent metal ion binding unequivocally excludes innersphere coordination to rna as an obligatory role. The target region must be free of secondary structure and have no proteinbinding. Immusol, inc, 3050 science park road, san diego, 92121, ca, usa.
Pdf antihbv hairpin ribozymemediated cleavage of target. It acts as an endonuclease that catalyzes a reversible sequencespecific cleavage reaction within a substrate rna 1619. The sequence requirements of the hairpin ribozyme have been elegantly determined by in vitro selection experiments and confirmed by mutagenesis 49. The active site is comprised of two stems each with an internal loop that forms a series of noncanonical base pairs. Hydroxyl groups essential for catalytic function of the hairpin. Ribozymes have been proposed as gene therapy agents against hiv1, although many fundamental questions about their mechanism of action remain unclear. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials. The hairpin ribozyme catalyzes efficient cleavage of an external rna substrate feldstein et al.
Among the small endonucleolytic ribozymes, the hairpin ribozyme possesses the unique feature of the internal equilibrium between cleavage and. After substrate cleavage and product dissociation, ribozyme recovers its ground state. Here we explore the structure and dynamics of key active site residues using more than 1. The hairpin ribozyme requires functional groups from ade38 to achieve efficient bond cleavage or ligation.
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. We have carried out hybrid quantummechanicalmolecular mechanical qmmm calculations to evaluate the energy along several. Singlemolecule fluorescence resonance energy transfer studies of freely diffusing hairpin ribozymes with different combinations of helical junction and loop elements reveal striking differences in their folding behavior. Like the hammerhead ribozyme it is found in rna satellites of plant viruses. Mar 15, 2002 the hairpin ribozyme presents an excellent case study for such a comparison. Metal ions play a passive role in the hairpin ribozyme. The hairpin ribozyme presents an excellent case study for such a comparison. Pdf probing nonselective cation binding in the hairpin. Residues a38 and g8 have been implicated as playing roles in general acid and base catalysis, respectively. Structure and function of the hairpin ribozyme sciencedirect. Ribozymes can be engineered to suppress the expression of genes, such as those that promote cancer or those from pathogenic viruses. Resultsanddiscussion mutations in the native sequence ofthe hairpin ribozyme and substrate fig. The hairpin ribozyme is a good model to explore this question because of the relatively simple secondary structure and the strong dependence of tertiary folding on the helical junction geometry.
Hairpin ribozyme lacking a substrate moiety, comprising at least six bases in helix 2 and able to basepair with a separate substrate rna, wherein the ribozyme comprises one or more bases 3 of helix 3 able to basepair with the substrate rna to form a helix 5 and wherein the said ribozyme can cleave andor ligate the separate rnas in trans. The hairpin ribozyme is a small, naturally occurring rna that catalyzes the reversible cleavage of rna substrates. The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic rna modification method that. To identify molecular features that contribute to catalysis, structures of position 38 base variants 2,6diaminopurine dap, 2aminopurine ap, cytosine cyt, and guanine gua were determined between 2. In this study, we have used fluorescence resonance energy transfer to analyze the conformation and. Current status and future prospects find, read and cite all the research you need on researchgate. Download fulltext pdf download fulltext pdf antihbv hairpin ribozymemediated cleavage of target rna in vitro article pdf available in world journal of gastroenterology 81 february 2002. Kinetics of tertiary structure formation in the hairpin ribozymesubstrate complex can be monitored by fret. Structure and activity of the hairpin ribozyme in its. The dominant tis delta virus hdv, and hairpin ribozymes catalyze the reactions of figure 1a and are of the class. The secondary structure of the vs ribozyme in the crystal conforms to the model derived from biochemical footprinting and mutagenesis 6, with the exception of junction 127 whose secondary structure had not been defined fig. Pdf hairpin ribozymes are small rna catalytic motifs naturally found in the satellite rnas of tobacco ringspot virus trsv, chicory yellow mottle. Evidence for the role of active site residues in the.
The catalytic mechanism of hairpin ribozyme studied by. Guanine 8 g8 and adenine 38 a38 have been identified as key participants in selfcleavage and ligation. Inhibition of tissue factor gene induction and activity. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. Protein factors may be involved in vivo or other, as. Among the small endonucleolytic ribozymes, the hairpin ribozyme possesses the unique feature of the internal equilibrium between cleavage and ligation being shifted toward ligation. 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. The hairpin ribozyme is a prominent member of small ribozymes since it does not require metal ions to achieve catalysis. For this reason we have explored the folding of the hairpin ribozyme, and its. Ligation of the hairpin ribozyme in cis induced by. Intracellular rna cleavage by the hairpin ribozyme.
Stability of hairpin ribozyme tertiary structure is governed by the interdomain junction. The hairpin ribozyme is an rnamotif that catalyzes rnaprocessing reactions essential forreplication of the satellite rnamolecules in which it is embedded. Evidence for the role of active site residues in the hairpin. The invention also includes a diagnostic assay for determining the presence of hpv16 in tissues based on pcr amplification. Innersphere coordinated metal ions are not required, as the inert metal ion complex cobalt hexammine promotes catalysis.
Recent structurefunction studies have begun to yield insights into the molecular bases of. One of these types seems to play a role in the folding and docking of the two domains and can be satisfied by a range of metal ions magnesium, calcium, strontium, manganese 11. We examined a series of six different ribozymes consisting of two, three and fourway junction variants, as well as corresponding constructs with one of. Two independent nmr structures have been determined for the hairpin ribozyme. Aug 05, 2003 the natural form of the hairpin ribozyme comprises two major structural elements. Efficacy and mode of action of hammerhead and hairpin. 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.
Tertiary structure formation in the hairpin ribozyme. 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. A fourway junction accelerates hairpin ribozyme folding. Ribozymes, or catalytic rnas, were first discovered in the laboratory of tom cech, at the university of colorado, in 1982. For comparison, a native allrna g8 hairpin ribozyme structure was refined to 2. All these catalytic rnas reversibly cleave the phosphodiester bond of substrate rna to generate 5. Adjacent arms a and b contain loops that include all but one of the nucleotides shown to be essential for catalytic activity, and the site of cleavageligation is. Few studies have compared directly the potential of different modified ribozyme species against a particular target. Comparative analysis of hairpin ribozyme structures and. The natural form of the hairpin ribozyme comprises two major structural elements.
The hairpin ribozyme against the u5 site recognizes a slightly longer sequence. The hairpin ribozyme consists of two loopcarrying duplexes called a and b that are adjacent arms of a fourway junction in its natural context in the viral rna. Hairpin ribozyme cleavage catalyzed by aminoglycoside. 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. In the configuration that is most commonly used for enzymological studies, nonessential satellite rna sequences are. Reactions were performed with 25 nmoll ribozyme and 100 nmoll substrate concentrations for the entire study. Download fulltext pdf download fulltext pdf antihbv hairpin ribozymemediated cleavage of target rna in vitro article pdf available in world journal of gastroenterology 81. Recent studies of the hairpin riboxyme have revealed a distinct catalytic mechanism for this small rna motif. Under solution conditions typically used in studies of the hairpin ribozyme i. Qmmm studies of hairpin ribozyme selfcleavage suggest. We find that the junctionbased form of the hairpin. The natural form of the hairpin ribozyme consists of a fourway rna junction of which the singlestranded loopcarrying helices are adjacent arms.
Intracellular application of hairpin ribozyme genes against. The hairpin ribozyme is a selfcleaving motif found in the negative strand of the satellite rna of some plant viruses. Ribozyme complexes can assemble in a number of ways. 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. The hairpin ribozyme catalyses sequencespecific cleavage of rna. Metal ion binding and the folding of the hairpin ribozyme. Folding and catalysis by the hairpin ribozyme sciencedirect. The hairpin ribozyme undergoes a sitespecific transesterification cleavage.
The hairpin ribozyme is indicated by upper case letters. 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. 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. Loopsize variation to probe a bent structure of a hairpin. The small catalytic rnas include the hammerhead, the hairpin or paperclip, hepatitis delta and vs rna. Comparison of ab initio, dft, and semiempirical qmmm. The hairpin ribozyme is a catalytic rna that functions in nature as a sitespecific ribonuclease and an rna ligase. The secondary structure prediction of the ribozyme is. Intracellular application of hairpin ribozyme genes against hepatitis b virus. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. May 20, 20 the hairpin ribozyme is an rnamotif that catalyzes rnaprocessing reactions essential forreplication of the satellite rnamolecules in which it is embedded. Inhibition of tissue factor gene induction and activity using. Helix 2 is fixed in length at 4 bases by the functional requirements of the hairpin ribozyme. The hairpin ribozyme is centered on a fourway helical junction, with arms sequentially labeled a, b, c, and d fig.
Pdf on sep 1, 1996, j m burke and others published hairpin ribozyme. Folding of the hairpin ribozyme in its natural conformation. Here we compare the relative abilities of hammerhead hhu5 and hairpin hpu5 ribozymes directed against a wellstudied target. Zhuoping cai and, ignacio tinoco, jr solution structure of loop a from the hairpin ribozyme from tobacco ringspot virus satellite. Wo1995031552a2 human papilloma virus inhibition by a. We have shown previously that the activity of the ribozyme is strongly influenced by the structure adopted by the junction. The hairpin ribozyme is a unique catalytic rna that achieves rate. Folding of the fourway rna junction of the hairpin ribozyme. A, substrate binds to a homogeneous ribozyme population in two different conformations, producing active and inactive ribozymesubstrate complexes. In its natural context, the ribozyme comprises four helices, two of which contain conserved formally unpaired loops, that are adjacent arms of a fourway rna junction. In this structure, the bases of loop a form a series of four noncanonical pairs resulting in an extended. Analysis of hairpin ribozyme base mutations in loops 2 and 4 and their effects on ciscleavage in vitro. Early studies of the ionic requirements for hairpin ribozyme cleavage showed that at least two types of ion were needed for efficient hairpin ribozyme cleavage.
Kinetic models for the hairpin ribozyme reactions, including two populations of ribozymesubstrate complexes. To identify g8 features that contribute to catalysis within the hairpin ribozyme active site, structures for five base variants were determined by xray crystallography in a resolution range between 2. The hairpin ribozyme is a small section of rna that can act as a ribozyme. The hairpin ribozyme is a small catalytic rna isolated from the minus strand of the tobacco ringspot virus satellite rna. Image showing the diversity of ribozyme structures. The active site of this natural rna results from the docking of two irregular helices.
We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed. The hairpin ribozyme is the minimal selfcleavage domain of the negative strand of the satellite rna of tobacco ringspot virus. We cloned hammerhead or hairpin ribozymes into a vector that was driven by the bacteriophage t 7 or the cytomegalovirus cmv immediate early rna. 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. Recent structurefunction studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme. Recent studies of the hairpin ribozyme have revealed a distinct catalytic mechanism for this small rna motif. Ribozyme can also catalyze the formation of peptide bond between adjacent amino acid by lowering the activation entropy. The characteristics and properties of catalytic rnas. His research team found that the ribosomal rna precursor from tetrahymena thermophila contained an intron, a nonencoding sequence that interrupts the gene, that was capable of excising itself, in vitro, without any protein or external energy source. This construct is derived from the work of feldstein and bruening 42 with reinforcement of helix 4, previously shown to enhance catalytic activity 26. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200fold.
Crystal structure of the vs ribozyme pubmed central pmc. The hairpin ribozyme, along with the hammerhead, axehead and newospora vs catalytic rnas, belongs to the family of small rna. These loops dock into each other to create an active site for catalysis. Here we present a systematic study of the folding behavior of hairpin ribozymes containing different combinations of loop and helical junction elements. A fourway junction accelerates hairpin ribozyme folding via.
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