Hairpin ribozyme
Encyclopedia
The hairpin ribozyme is a small section of RNA
that can act as an enzyme known as a ribozyme
. Like the hammerhead ribozyme
it is found in RNA satellite
s of plant viruses. It was first identified in the minus strand of the tobacco ringspot virus (TRSV) satellite RNA where it catalyzes
self-cleavage and joining (ligation
) reactions
to process the products of rolling circle virus replication
into linear and circular satellite RNA molecules. The hairpin ribozyme is similar to the hammerhead ribozyme in that it does not require a metal ion for the reaction.
. At the end of the replication cycle, these large intermediates of satellite RNA replication are processed down to unit length molecules (circular or linear) before they can be packaged by viruses and carried to other cells for further rounds of replication.
Smaller artificial versions of the hairpin ribozyme have been developed to enable a more detailed experimental analysis of the molecule. This is a commonly used strategy for separating those parts of a self-processing RNA molecule that are essential for the RNA processing reactions from those parts which serve unrelated functions. Through this process, a 50 nucleotide minimal catalytic domain and a 14 nucleotide substrate were identified. Using these artificially derived sequences, a trans-acting ribozyme was developed that can catalyze the cleavage of multiple substrate molecules. This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes.
used in both RNA and DNA. Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes.
that includes two domains, each consisting of two short base pair
ed helices separated by an internal loop. Domain A (helix 1 - loop A - helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 - loop B - helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These domains must interact with one another in order for catalysis to occur.
When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength
, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin. In order for catalysis to occur, the two domains lie parallel to one another in a fold that resembles a paperclip. In various publications, this RNA has been termed either the "paperclip" or "hairpin" ribozyme. Despite the fact that the former name has proven to be more accurate, the latter has become the commonly accepted nomenclature. In the laboratory, a functional interaction between the two domains is promoted by the addition of cations
, whose positive charge suffices to overcome the electrostatic repulsion
of the negatively charged RNA backbone. In nature, association of the two domains is assisted through a combination of metal ions (including Mg2+) and the presence of two additional helical domains that are not present in the minimal ribozyme-substrate complex but serve to promote proper three-dimensional folding. These additional domains stack upon helices 2 and 3, thereby promoting the association of the two functional domains through what is termed a four-way helical junction.
The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy
, covalent crosslinking
, NMR
analysis and x-ray crystallography
. These studies have been facilitated by the ability of the functional complex to self-assemble from segments made by solid phase chemical RNA synthesis
, permitting the incorporation of a wide variety of modified nucleotides that are not naturally found in RNA. Together, the results of these experiments present a highly congruent picture of the catalytic cycle
, i.e. how the hairpin ribozyme binds its substrate, folds into a specific three-dimensional structure, catalyzes the reaction, and releases the product(s) of the reaction.
ing within helices 1 and 2.
One area of particular interest has been the development of hairpin ribozymes for potential therapeutic use, for example by preventing the replication of pathogenic viruses. Antiviral hairpin ribozymes have been generated and expressed within mammalian cells, and cells expressing different engineered ribozymes have been shown to be resistant to infection by HIV-1, hepatitis B, and Sindbis virus
.
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
that can act as an enzyme known as a ribozyme
Ribozyme
A ribozyme is an RNA molecule with a well defined tertiary structure that enables it to catalyze a chemical reaction. Ribozyme means ribonucleic acid enzyme. It may also be called an RNA enzyme or catalytic RNA. Many natural ribozymes catalyze either the hydrolysis of one of their own...
. Like the hammerhead ribozyme
Hammerhead ribozyme
Hammerhead RNAs are RNAs that self-cleave via a small conserved secondary structural motif termed a hammerhead because of its shape. Most hammerhead RNAs are subsets of two classes of plant pathogenic RNAs: the satellite RNAs of RNA viruses and the viroids. The self-cleavage reactions, first...
it is found in RNA satellite
Satellite (biology)
A satellite is a subviral agent composed of nucleic acid that depends on the co-infection of a host cell with a helper or master virus for its multiplication. When a satellite encodes the coat protein in which its nucleic acid is encapsidated it is referred to as a satellite virus...
s of plant viruses. It was first identified in the minus strand of the tobacco ringspot virus (TRSV) satellite RNA where it catalyzes
Catalysis
Catalysis is the change in rate of a chemical reaction due to the participation of a substance called a catalyst. Unlike other reagents that participate in the chemical reaction, a catalyst is not consumed by the reaction itself. A catalyst may participate in multiple chemical transformations....
self-cleavage and joining (ligation
Chemical ligation
Chemical ligation is a set of techniques used for creating long peptide or protein chains. It is the second step of a convergent approach. First, smaller peptides containing 30-50 amino acids are prepared by conventional chemical peptide synthesis. Then, they are completely deprotected...
) reactions
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
to process the products of rolling circle virus replication
Rolling circle replication
Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids...
into linear and circular satellite RNA molecules. The hairpin ribozyme is similar to the hammerhead ribozyme in that it does not require a metal ion for the reaction.
Biological function
The hairpin ribozyme is an RNA motif that catalyzes RNA processing reactions essential for replication of the satellite RNA molecules in which it is embedded. These reactions are self-processing, i.e. a molecule rearranging its own structure. Both cleavage and end joining reactions are mediated by the ribozyme motif, leading to a mixture of interconvertible linear and circular satellite RNA molecules. These reactions are important for processing the large multimeric RNA molecules that are generated by rolling circle replicationRolling circle replication
Rolling circle replication describes a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the genomes of bacteriophages, and the circular RNA genome of viroids...
. At the end of the replication cycle, these large intermediates of satellite RNA replication are processed down to unit length molecules (circular or linear) before they can be packaged by viruses and carried to other cells for further rounds of replication.
Natural and artificial versions of the hairpin ribozyme
The hairpin ribozyme has been identified in only 3 naturally occurring sequences:- satellite RNA of tobacco ringspot virusTobacco ringspot virusTobacco ringspot virus is a plant pathogenic virus in the plant virus family Comoviridae. It is the type species of the Comoviridae Genus Nepovirus. Nepoviruses are be transmitted between plants by nematodes...
(sTRSV) - satellite RNA of chicory yellow mottle virus (sCYMV)
- satellite RNA of arabis mosaic virusArabis mosaic virusArabis mosaic virus is a plant pathogenic virus of the family Comoviridae.-External links:**...
(sARMV)
Smaller artificial versions of the hairpin ribozyme have been developed to enable a more detailed experimental analysis of the molecule. This is a commonly used strategy for separating those parts of a self-processing RNA molecule that are essential for the RNA processing reactions from those parts which serve unrelated functions. Through this process, a 50 nucleotide minimal catalytic domain and a 14 nucleotide substrate were identified. Using these artificially derived sequences, a trans-acting ribozyme was developed that can catalyze the cleavage of multiple substrate molecules. This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes.
Reaction chemistry
In common with several other ribozymes and protein ribonucleases, the cleavage reaction of the hairpin ribozyme generates RNA fragments with termini consisting of a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The ligation reaction appears to be a simple reversal of cleavage, i.e. covalent joining of RNA fragments ending with a 2',3'-cyclic phosphate and a 5'-hydroxyl group to generate the ordinary 3'-5' phosphodiester linkagePhosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...
used in both RNA and DNA. Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes.
Structure
The minimal hairpin ribozyme-substrate complex folds into a secondary structureNucleic acid secondary structure
The secondary structure of a nucleic acid molecule refers to the basepairing interactions within a single molecule or set of interacting molecules, and can be represented as a list of bases which are paired in a nucleic acid molecule....
that includes two domains, each consisting of two short base pair
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
ed helices separated by an internal loop. Domain A (helix 1 - loop A - helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 - loop B - helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These domains must interact with one another in order for catalysis to occur.
When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength
Ionic strength
The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic compounds, when dissolved in water, dissociate into ions. The total electrolyte concentration in solution will affect important properties such as the dissociation or the solubility of different salts...
, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin. In order for catalysis to occur, the two domains lie parallel to one another in a fold that resembles a paperclip. In various publications, this RNA has been termed either the "paperclip" or "hairpin" ribozyme. Despite the fact that the former name has proven to be more accurate, the latter has become the commonly accepted nomenclature. In the laboratory, a functional interaction between the two domains is promoted by the addition of cations
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
, whose positive charge suffices to overcome the electrostatic repulsion
Electrostatics
Electrostatics is the branch of physics that deals with the phenomena and properties of stationary or slow-moving electric charges....
of the negatively charged RNA backbone. In nature, association of the two domains is assisted through a combination of metal ions (including Mg2+) and the presence of two additional helical domains that are not present in the minimal ribozyme-substrate complex but serve to promote proper three-dimensional folding. These additional domains stack upon helices 2 and 3, thereby promoting the association of the two functional domains through what is termed a four-way helical junction.
The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy
Fluorescence spectroscopy
Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit...
, covalent crosslinking
Cross-link
Cross-links are bonds that link one polymer chain to another. They can be covalent bonds or ionic bonds. "Polymer chains" can refer to synthetic polymers or natural polymers . When the term "cross-linking" is used in the synthetic polymer science field, it usually refers to the use of...
, NMR
Nuclear magnetic resonance
Nuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
analysis and x-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
. These studies have been facilitated by the ability of the functional complex to self-assemble from segments made by solid phase chemical RNA synthesis
Oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
, permitting the incorporation of a wide variety of modified nucleotides that are not naturally found in RNA. Together, the results of these experiments present a highly congruent picture of the catalytic cycle
Catalytic cycle
A catalytic cycle in chemistry is a term for a multistep reaction mechanism that involves a catalyst . The catalytic cycle is the main method for describing the role of catalysts in biochemistry, organometallic chemistry, materials science, etc. Often such cycles show the conversion of a...
, i.e. how the hairpin ribozyme binds its substrate, folds into a specific three-dimensional structure, catalyzes the reaction, and releases the product(s) of the reaction.
Targeted RNA cleavage and antiviral activity
Hairpin ribozymes have been modified in such a way that they can be used to target cleavage of other RNA molecules. This is possible because much of the substrate specificity of the hairpin ribozyme results from simple Watson-Crick base pairBase pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
ing within helices 1 and 2.
One area of particular interest has been the development of hairpin ribozymes for potential therapeutic use, for example by preventing the replication of pathogenic viruses. Antiviral hairpin ribozymes have been generated and expressed within mammalian cells, and cells expressing different engineered ribozymes have been shown to be resistant to infection by HIV-1, hepatitis B, and Sindbis virus
Sindbis virus
Sindbis Virus is a member of the Togaviridae family, in the alphavirus subfamily. The virus was first isolated in 1952 in Cairo, Egypt. The virus is transmitted by mosquitoes SINV causes sindbis fever in humans and the symptoms include arthralgia, rash and malaise...
.