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. In the configuration that is most commonly used for enzymological studies, nonessential satellite rna sequences are simply deleted leaving ribozyme. The hairpin ribozyme fig 3b has also been split into substrate and catalytic core. Interdomain crosslinking and molecular modelling of the. Here, we report the in vitro evolution of an improved rna polymerase ribozyme that is able to synthesize structured functional rnas, including aptamers and. Apr 12, 2001 the hairpin ribozyme catalyses sequencespecific cleavage of rna.
Residues a38 and g8 have been implicated as playing roles in general acid and base catalysis, respectively. Dna aptamermediated regulation of the hairpin ribozyme by. The hairpin ribozyme is a member of a family of small rna endonucleases, which includes hammerhead, human hepatitis delta virus, neurospora vs, and the. Rna molecules and design of ribozymes are mainly theoretical. Proteopedia is hosted by the ispc at the weizmann institute of science in israel. Structural basis for substrate helix remodeling and. Proteindependent ribozymes report molecular interactions. The active form of the hairpin ribozyme is brought about by the interaction of two formally unpaired loops. Base and sugar requirements for rna cleavage of essential.
If it is possible to combine and to control cleavage and ligation activity, a hairpinderived twin ribozyme might be a potential tool for rna manipulation in vitro, with the possibility in future of applying a similar method in vivo. All these catalytic rnas reversibly cleave the phosphodiester bond of substrate rna to generate 5. Evidence for the role of active site residues in the. Resultsanddiscussion mutations in the native sequence ofthe hairpin ribozyme and substrate fig.
They may catalyze selfcleavage intramolecular or incis catalysis as well as the cleavage of external substrates intermolecular or intrans catalysis ohkawa, 1995. It is one of several catalytic rnas known to occur in nature. A berzalherranz, s joseph, b m chowrira, s e butcher, and j m burke department of microbiology and molecular genetics, markey center for molecular genetics, university of vermont, burlington 05405. Two clinical trials with hammerhead ribozyme for treatment of hiv1 have been advanced in us. Hairpin ribozyme cleavage catalyzed by aminoglycoside. Although activity can be obtained in molecules lacking this junction, the junction is important in the promotion of the folded state of the ribozyme under physiological conditions, at a rate that is. The hairpin ribozyme accelerates a phosphoryl transfer reaction without catalytic participation of divalent metal ions. Structure and function of the hairpin ribozyme sciencedirect. The hairpin ribozyme is a small catalytic rna that has been reengineered resulting in a number of variants with extended or even new functions. To overcome its sequence dependence, we recombined traits evolved separately in different. Hdv, hairpin, and neurospora varkud satellite vs ribozymes are small rnas of 50150 nucleotides that perform site. Rna double cleavage by a hairpinderived twin ribozyme. We have used a chemical synthesis approach to study the requirements of hairpin rna cleavage for sugar and base moieties in residues of internal loop b, an essential region in one of the two ribozyme domains.
A hairpin ribozyme inhibits expression of diverse strains. Found in bacteria, lower eukaryotes and higher plants. The hammerhead ribozyme is an rna motif that catalyzes reversible cleavage and ligation reactions at a specific site within an rna molecule. Secondary structure model of the hairpin ribozyme construct used in this work. The hepatitis delta virus hdv ribozyme is a noncoding rna found in the hepatitis delta virus that is necessary for viral replication and is the only known human virus that utilizes ribozyme activity to infect its host. The target region must be free of secondary structure and have no proteinbinding sites. 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. A fourway junction accelerates hairpin ribozyme folding via a discrete intermediate elliot tan, timothy j. Like the hammerhead ribozyme it is found in rna satellites of plant viruses. The active conformation of the hairpin ribozyme has been. A critical event in the origin of life is thought to have been the emergence of an rna molecule capable of replicating a primordial rna genome. Typically, ribozymes possess nucleotide sequences that are complementary to a target rna of interest. Mar 02, 2017 hairpin ribozyme design employs the same approach except that the catalytic core is larger 34 nucleotides and the ribozyme targeting domains require more specificity.
The active site of this natural rna results from the docking of two irregular helices. We have determined the crystal structure of a hairpinribozymeinhibitor complex at a resolution of 2. Selection of targets and the most efficient hairpin ribozymes for. The ribozyme acts to process the rna transcripts to unit lengths in a selfcleavage reaction during replication of the hepatitis delta virus, which is thought to propagate by. 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. The km value for the monosubstituted ribozyme with a 2,4difluorobenzene is 309 nm1, the corresponding kcat is 2. The substratebinding domain has been optimized to reduce misfolding 15, 22. After substrate cleavage and product dissociation, ribozyme recovers its ground state. The hairpin ribozyme is a small selfcleaving rna that can be engineered for rna cleavage in trans and has potential as a therapeutic agent.
Hairpin ribozyme design employs the same approach except that the catalytic core is larger 34 nucleotides and the ribozyme targeting domains require more specificity. Biochemical work has pinpointed specific bases that affect the fold and function by mechanisms that alter longdistance contacts to destabilize the catalytic core, and by identification of active site variants that directly alter the chemical steps of catalysis 1820. The recently determined crystal structures of hammerhead ribozymes reveal how a. Hairpins recognize the sequence nnyngucnnnnnn, where n is any nucleotide and y is a pyrimidine. A u39c mutation has been included which has been shown to increase the activity of the ribozyme 8. Ribozymes are derived from the wildtype transacting ribozyme with the following modifications. Crystal structure of a hairpin ribozymeinhibitor complex.
Essential nucleotide sequences and secondary structure. Ribozymecatalysed rna synthesis using triplet building. 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. In the case of the hairpin ribozyme, pressure slowed down the selfcleavage reaction on the basis that the. In vitro selection of active hairpin ribozymes by sequential. Although all extant life relies on protein enzymes to accomplish these tasks, life in the ancestral rna world would have used only rna enzymes. Analogous work is under way to establish the active structure of the hammerhead ribozyme.
It serves as a model system for research on the structure and properties of rna, and is used for targeted rna cleavage experiments, some with proposed therapeutic applications. It is of particular biochemical interest because, unlike classic ribozymes, such as the group i intron, it appears not to employ metal ions as catalytic cofactors. Pdf recent experimental work on the hairpin and hammerhead ribozymes. Amplification of rna by an rna polymerase ribozyme pnas. 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. Proceedings of the national academy of sciences of the united. Group i and group ii intron splicing ribozymes rnase p hammerhead ribozyme hairpin ribozyme ribozome 8.
Group i introns are also found inserted into genes of a wide varietyof bacteriophages of gram. Kinetic models for the hairpin ribozyme reactions, including two populations of ribozymesubstrate complexes. May 20, 20 the hairpin ribozyme is an rnamotif that catalyzes rnaprocessing reactions essential forreplication of the satellite rnamolecules in which it is embedded. Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. Ribozymecatalyzed transcription of an active ribozyme. Articles the solventprotected core of the hairpin ribozymesubstrate complex ken j. Thepercent activity values arethe cleavage rates for the mutation relative to those for the native sequence. In a natural molecule, these are present on two adjacent arms of a fourway junction.
Lilley, and taekjip ha department of physics and center for biophysics and computational biology, university of illinois at urbanachampaign, urbana, il 61801. Feb 26, 2019 one main example of a large ribozyme is the first one to be discovered, which is the tetrahymena group i intron. The hairpin ribozyme is one of the most well studied small rna enzymes. The therapeutic potential of ribozymes blood american. The minimum catalytic center of s trsv was identified, biochemically characterized and named the hairpin ribozyme 1, 2. The hairpin ribozyme is one of four known natural catalytic rnas that carry out sequencespecific cleavage of rna. Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. 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. The hammerhead ribozymecatalyzed reaction, unlike the formally identical nonenzymatic alkaline cleavage of rna, is a highly sequencespecific cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate per molecule of enzyme per minute at ph 7. A fourway junction accelerates hairpin ribozyme folding. One main example of a large ribozyme is the first one to be discovered, which is the tetrahymena group i intron. A disubstituted hammerhead ribozyme carrying one of each. 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 an rnamotif that catalyzes rnaprocessing reactions essential forreplication of the satellite rnamolecules in which it is embedded.
The hairpin ribozyme is a wellstudied example of a highly structured functional rna that exhibits pronounced heterogeneity. The catalytic activity of the hairpin ribozyme extends to both cleavage and ligation reactions. A, substrate binds to a homogeneous ribozyme population in two different conformations, producing active and inactive ribozymesubstrate complexes. Ribozymes can be engineered to suppress the expression of genes, such as those that promote cancer or those from pathogenic viruses. Exploring ribozyme conformational changes with xray. In vitro selection of active hairpin ribozymes by sequential rnacatalyzed cleavage and ligation reactions. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Structural basis of specific trna aminoacylation by a.
Previously, replacement of g8 by a series of nucleobase variants showed little effect on interdomain docking, but a 3. Articles the solventprotected core of the hairpin ribozyme. Burke department of microbiology and molecular genetics, markey center for molecular genetics, university of vermont. The minimum sequence for catalytic activity was found to be a 50. These data further support the potential of this hairpin ribozyme as a therapeutic agent for hiv1. Further, the hairpin ribozyme is also an efficient ligase 25,26.
Cleavage site nucleotides are shown in cyan, catalytic nucleotides in red, and loop a and loop b nucleotides in green and orange, respectively. The small selfcleaving rnas, including the hairpin, hammerhead, hepatitis delta and neurospora vs ribozymes, catalyze a reversible cleavage reaction that generates 2. 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. Structural basis of specific trna aminoacylation by a small. Ribozyme complexes can assemble in a number of ways. Whereas the reaction products from family members are similar. Following initial identification of the minimum catalytic sequence, we identified a transcatalytic reaction and biochemically characterized this reaction.
Heterogeneous folding and function of small rna motifs. 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 is a 50nucleotide rna enzyme of unknown threedimensional structure. This is an advanced enzymology book and discusses most of the classical and modern aspects of enzymology in detail. Water in the active site of an allrna hairpin ribozyme. Dna aptamermediated regulation of the hairpin ribozyme by human. The hairpin ribozyme requires functional groups from ade38 to achieve efficient bond cleavage or ligation.
Herein we used raman crystallography to directly measure pka values for the ade38 n1 imino group of a hairpin ribozyme in distinct conformational states. Examples of small ribozymes include the hammerhead, the hairpin, the hepatitis. Ribozymes are active in vitro in cellfree systems and in living cells. The recently determined crystal structures of hammerhead ribozymes reveal how a small rna motif can fold up into a con. 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. Transacting hairpin ribozyme constructs used in this study. Recent studies of the hairpin riboxyme have revealed a distinct catalytic mechanism for this small rna motif. Here we explore the structure and dynamics of key active site residues using more than 1. M for a minimal ribozyme with the sequence derived from. Here we describe the evolution and engineering of an rna polymerase ribozyme capable of synthesizing rnas of up to 95 nucleotides in length. Characterization of hairpin ribozyme reactions springerlink. These include a protocol for ex vivo transduction and infusion of autologous tlymphocytes from infected individuals using murine vectors producing a hairpin ribozyme directed against the u5 leader sequence of hivwong and a protocol for the. Darwinian life requires the ability to replicate genotypes and express phenotypes.
Reconstitution of hairpin ribozyme activity following. Recent studies of the hairpin ribozyme have revealed a distinct catalytic mechanism for this small rna motif. Group i intron ribozymes constitute one of the main classes of ribozymes. It was first identified in the minus strand of the tobacco ringspot virus trsv satellite rna where it catalyzes selfcleavage and joining ligation reactions to process the products of rolling circle virus replication into linear and circular satellite rna molecules. Examples of ribozymes include the hammerhead ribozyme, the vs ribozyme, leadzyme and the hairpin ribozyme. A crystal structure of the precatalytic active site of the hairpin ribozyme pdb id. Evidence for the role of active site residues in the hairpin. The underlined target bases nguc are not basepaired with the ribozyme. Pentacytidine linker joining ribozyme and substrate is indicated by broken lines.
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. The hairpin ribozyme requires functional group contributions from g8 to assist in phosphodiester bond cleavage. The hairpin ribozyme catalyses sequencespecific cleavage of rna. The hairpin ribozyme is a small section of rna that can act as a ribozyme. 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. Structural basis for substrate helix remodeling and cleavage. Grant rafter, beyond disney page 280 the variety of angles in the course, from slow bends to hairpins, will both test your skill as a driver and allow you the opportunity to pass other carts.
The ribozyme recognizes sequences either side of a substrate nuh site by means of the flanking arms. 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. Innersphere coordinated metal ions are not required, as the inert metal ion complex cobalt hexammine promotes catalysis. 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. Ribozyme, or rna enzyme, is a rna molecule that act as enzymes, often found to catalyze cleavage of either its own or other rnas due to their complex secondary structures and hairpin hammer head active centres, rnas could act as a catalyst and this idea was proposed by carl woese et al, thomas r cech and sidney altman were the first to discover ribozyme. Scott and aaron klug the hammerhead rna is a small catalytic rna found in a number of rna virus genomes and viruslike rnas. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains a and b might fold and approach each.
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