Hammerhead ribozyme
Encyclopedia
Hammerhead RNAs are RNA
s 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 RNA
s of RNA viruses and the viroids. The self-cleavage reactions, first reported in 1986, are part of a rolling circle replication mechanism. The hammerhead sequence is sufficient for self-cleavage and acts by forming a conserved three-dimensional tertiary structure.
enzymes, the hammerhead RNA itself is not a true enzyme
in its natural state, as it cannot catalyze multiple turnovers.
In vitro
hammerhead constructs can be engineered such that they consist of two RNA strands. The strand that gets cleaved can be supplied in excess, and multiple turnover can be demonstrated and shown to obey Michaelis-Menten kinetics
, typical of protein enzyme kinetics
. Such constructs are typically employed for in vitro experiments, and the term "hammerhead RNA" has become in practice synonymous with the more frequently used "hammerhead ribozyme
".
The minimal hammerhead ribozyme sequence that is catalytically active consists of three base-paired stems flanking a central core of 15 conserved (mostly invariant) nucleotides, as shown. The conserved central bases, with few exceptions, are essential for ribozyme’s catalytic activity. Such hammerhead ribozyme constructs exhibit a turnover rate (kcat
) of about 1 molecule/minute and a Km
on the order of 10 nanomolar.
The hammerhead ribozyme is arguably the best-characterized ribozyme. Its small size, thoroughly-investigated cleavage chemistry, known crystal structure, and its biological relevance make the hammerhead ribozyme particularly well-suited for biochemical and biophysical investigations into the fundamental nature of RNA catalysis.
Hammerhead ribozymes may play an important role as therapeutic agents; as enzymes which tailor defined RNA sequences, as biosensor
s, and for applications in functional genomics
and gene discovery.
, the mechanism employed does not in fact involve the addition of water
. Rather, the cleavage reaction is simply an isomerization that consists of rearrangement of the linking phosphodiester bond. It is the same reaction, chemically, that occurs with random base
-mediated RNA degradation, except that it is highly site-specific and the rate is accelerated 10,000-fold or more.
2’-hydroxyl proton from the 2’-oxygen, which then becomes the attacking nucleophile
in an “in-line” or SN2(P)-like reaction, although it is not known whether this proton is removed prior to or during the chemical step of the hammerhead cleavage reaction. (The cleavage reaction is technically not bimolecular, but behaves in the same way a genuine SN2(P) reaction does; it undergoes inversion of configuration subsequent to forming an associative transition-state
consisting of a pentacoordinated oxyphosphrane.) The attacking and leaving group
oxygens will both occupy the two axial positions in the trigonal bipyramidal transition-state structure as is required for an SN2-like reaction mechanism.
The 5’-product, as a result of this cleavage reaction mechanism, possesses a 2’,3’-cyclic phosphate terminus, and the 3’-product possesses a 5’-OH terminus, as with nonezymatic alkaline cleavage of RNA. The reaction is therefore, in principle, reversible, as the scissile phosphate remains a phosphodiester, and may thus act as a substrate for hammerhead RNA-mediated ligation without a requirement for ATP
or a similar exogenous energy source. The hammerhead ribozyme-catalyzed reaction, unlike the formally identical non-enzymatic alkaline cleavage of RNA, is a highly sequence-specific cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate per molecule of enzyme per minute at pH 7.5 in 10 mM Mg2+ (so-called “standard reaction conditions” for the minimal hammerhead RNA sequence), depending upon the sequence of the particular hammerhead ribozyme construct measured. This represents an approximately 10,000-fold rate enhancement over the nonezymatic cleavage of RNA.
, in the sense that they were assumed to require the presence of divalent cations, such as Mg2+
, for both folding and catalysis. It was presumed that hexahydrated magnesium
ions, which exist in equilibrium with magnesium hydroxide
, could play the roles of general acid and general base, in a way analogous to those played by two histidines in RNase A. An additional role for divalent metal ions has also been proposed in the form of electrostatic stabilization of the transition-state
.
and hairpin ribozyme
, do not require the presence of metal ions for catalysis, provided a sufficiently high concentration of monovalent
cation is present to permit the RNA to fold. This discovery suggested that the RNA itself, rather than serving as an inert, passive scaffold for the binding of chemically active divalent metal ions, is instead itself intimately involved in the chemistry of catalysis. The latest structural results, described below, indeed confirm that two invariant nucleotides, G12 and G8, are positioned consistent with roles as the general base and general acid in the hammerhead cleavage reaction.
Strictly speaking, therefore, the hammerhead ribozyme cannot be a metallo-enzyme.
The minimal hammerhead sequence that is required for the self-cleavage reaction includes approximately 13 conserved or invariant "core" nucleotides, most of which are not involved in forming canonical Watson-Crick base-pairs
. The core region is flanked by Stems I, II and III, which are in general made of canonical Watson-Crick base-pairs
but are otherwise not constrained with respect to sequence. The catalytic turnover rate of minimal hammerhead ribozymes is ~ 1/min (a range of 0.1/min to 10/min is commonly observed, depending upon the nonconserved sequences and the lengths of the three helical stems). Much of the experimental work carried out on hammerhead ribozymes has used a minimal construct.
The minimal hammerhead ribozyme has been exhaustively studied by biochemists and enzymeologists as well as by X-ray crystallographers, NMR spectroscopists, and other practitioners of biophysical techniques. The first detailed three-dimensional structural information for a hammerhead ribozyme appeared in 1994 in the form of an X-ray crystal structure of a hammerhead ribozyme bound to a DNA substrate analogue, published in Nature
by Pley, Flaherty and McKay. Subsequently, an all-RNA minimal hammerhead ribozyme structure was published by Scott, Finch and Klug in Cell
in early 1995.
The minimal hammerhead ribozyme is composed of three base paired helices, separated by short linkers of conserved sequence as shown in the crystal structure
. These helices are called I, II and III. The conserved uridine-turn links helix I to helix II and usually contains the sequence CUGA. Helix II and III are linked by a sequence GAAA. The cleavage reaction occurs between helix III and I, and is usually a C.
The structure of a full length ribozyme shows that there are extensive interactions between the loop of stem II and stem I.
Hammerhead ribozymes can be divided into three classes according to which of the three stems is formed from the 5' and 3' end of the sequence region. If stem III is formed from the 5' and 3' most parts of the sequence they are known as class III.
For example, the invariant core residues G5, G8, G12 and C3 in the minimal hammerhead ribozyme were each observed to be so fragile that changing even a single exocyclic functional group on any one of these nucleotides results in a dramatic reduction or abolition of catalytic activity, yet few of these appeared to form hydrogen bonds involving the Watson-Crick faces of these nucleotide bases in any of the minimal hammerhead structures, apart from a G-5 interaction in the product structure.
A particularly striking and only recently observed example consisted of G8 and G12, which were identified as possible participants in acid/base catalysis. Once it was demonstrated that the hammerhead RNA does not require divalent metal ions for catalysis, it gradually became apparent that the RNA itself, rather than passively bound divalent metal ions, must play a direct chemical role in any acid-base chemistry in the hammerhead ribozyme active site. It was however completely unclear how G12 and G8 could accomplish this, given the original structures of the minimal hammerhead ribozyme.
Other concerns included an NOE between U4 and U7 of the cleaved hammerhead ribozyme that had also been observed during NMR characterization, which suggested that these nucleotide bases must approach one another closer than about 6 Å, although close approach of U7 to U4 did not appear to be possible from the crystal structure. Finally, as previously discussed, the attacking nucleophile in the original structures, the 2’-OH of C17, was not in a position amenable to in-line attack upon the adjacent scissile phosphate.
Perhaps most worrisome were experiments that suggested the A-9 and scissile phosphates must come within about 4 Å of one another in the transition-state, based upon double phosphorothioate substitution and soft metal ion rescue experiments; the distance between these phosphates in the minimal hammerhead crystal structure was about 18 Å, with no clear mechanism for close approach if the Stem II and Stem I A-form helices were treated as rigid bodies. Taken together, these results appeared to suggest that a fairly large-scale conformational change must have take place in order to reach the transition-state within the minimal hammerhead ribozyme structure.
For these reasons, the two sets of experiments (biochemical vs. crystallographic) appeared not only to be at odds, but to be completely and hopelessly irreconcilable, generating a substantial amount of discord in the field. No compelling evidence for dismissing either set of experimental results was ever made successfully, although many claims to the contrary were made in favor of each.
In 2006 a 2.2 Å resolution crystal structure of the full-length hammerhead ribozyme was obtained. This new structure (shown on the right) appears to resolve the most worrisome of the previous discrepancies. In particular, C17 is now positioned for in-line attack, and the invariant residues C3, G5, G8 and G12 all appear involved in vital interactions relevant to catalysis. Moreover, the A9 and scissile phosphates are observed to be 4.3 Å apart, consistent with the idea that, when modified, these phosphates could bind a single thiophilic metal ion. The structure also reveals how two invariant residues, G-12 and G-8, are positioned within the active site consistent with their previously proposed role in acid/base catalysis. G12 is within hydrogen bonding distance to the 2’–O of C17, the nucleophile in the cleavage reaction, and the ribose of G8 hydrogen bonds to the leaving group 5’-O. (see below), while the nucleotide base of G8 forms a Watson-Crick pair with the invariant C3. This arrangement permits one to suggest that G12 is the general base in the cleavage reaction, and that G8 may function as the general acid, consistent with previous biochemical observations. G5 hydrogen bonds to the furanose oxygen of C17, helping to position it for in-line attack. U4 and U7, as a consequence of the base-pair formation between G8 and C3, are now positioned such that an NOE between their bases is easily explained.
The crystal structure of the full-length hammerhead ribozyme thus clearly addresses all of the major concerns that appeared irreconcilable with the previous crystal structures of the minimal hammerhead ribozyme.
The tertiary interactions in the full-length hammerhead ribozyme stabilize what strongly appears to be the active conformation. The nucleophile, the 2'-oxygen of the cleavage-site nucleotide, C17, is aligned almost perfectly for an in-line attack (the SN2(P) reaction). G12 is positioned within hydrogen bonding distance of this nucleophile, and therefore would be able to abstract a proton from the 2'-oxygen if G12 itself becomes deprotonated. The 2'-OH of G8 forms a hydrogen bond to the 5'-leaving group oxygen, and therefore potentially may supply a proton as negative charge accumulates on the 5'-oxygen of the ribose of A1.1.
The most likely explanation is then that G12, in the deprotonated form, is the general base, and the ribose of G8 is the general acid. The apparent kinetic pKa of the hammerhead ribozyme is 8.5, whereas the pKa of guanosine is about 9.5. It is possible that the pKa of G12 is perturbed from 9.5 to 8.5 in the hammerhead catalytic core; this hypothesis is currently the subject of intense investigation.
If the invariant G8 is changed to C8, hammerhead catalysis is abolished. However, a G8C + C3G double-mutant that maintains the G8-C3 base pair found in the full-length hammerhead restores most of the catalytic activity. The 2'-OH of G8 has also been observed to be essential for catalysis; replacement of G8 with deoxyG8 greatly reduces the rate of catalysis, suggesting the 2'-OH is indeed crucial to the catalytic mechanism.
The close approach of the A9 and scissile phosphates requires the presence of a high concentration of positive charge. This is probably the source of the observation that divalent metal ions are required at low ionic strength, but can be dispensed with at higher concentrations of monovalent cations.
The reaction thus likely involves abstraction of the 2'-proton from C17, followed by nucleophilic attack upon the adjacent phosphate. As the bond between the scissile phosphorus and the 5'-O leaving group begins to break, a proton is supplied from the ribose of G8, which then likely reprotonates at the expense of a water molecule observed to hydrogen bond to it in the crystal structure.
. Work is also underway to find out whether they could be used to engineer HIV
resistant lines of T-cells.
The therapeutic use of trans-cleaving hammerhead ribozymes has been severely hampered by its low-level activity in vivo. The true catalytic potential of trans-cleaving hammerhead ribozymes may be recouped in vivo and therapeutic derivatives are likely to complement other nucleic acid hybridizing therapeutic strategies. Already there are hammerhead ribozymes which are close to clinical application.
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
s 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 RNA
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 RNA viruses and the viroids. The self-cleavage reactions, first reported in 1986, are part of a rolling circle replication mechanism. The hammerhead sequence is sufficient for self-cleavage and acts by forming a conserved three-dimensional tertiary structure.
Autocatalyst or enzyme?
In the natural state, a hammerhead RNA motif is a single strand of RNA, and although the cleavage is autocatalytic and takes place in the absence of proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
enzymes, the hammerhead RNA itself is not a true enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
in its natural state, as it cannot catalyze multiple turnovers.
In vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
hammerhead constructs can be engineered such that they consist of two RNA strands. The strand that gets cleaved can be supplied in excess, and multiple turnover can be demonstrated and shown to obey Michaelis-Menten kinetics
Michaelis-Menten kinetics
In biochemistry, Michaelis–Menten kinetics is one of the simplest and best-known models of enzyme kinetics. It is named after German biochemist Leonor Michaelis and Canadian physician Maud Menten. The model takes the form of an equation describing the rate of enzymatic reactions, by relating...
, typical of protein enzyme kinetics
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
. Such constructs are typically employed for in vitro experiments, and the term "hammerhead RNA" has become in practice synonymous with the more frequently used "hammerhead 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...
".
The minimal hammerhead ribozyme sequence that is catalytically active consists of three base-paired stems flanking a central core of 15 conserved (mostly invariant) nucleotides, as shown. The conserved central bases, with few exceptions, are essential for ribozyme’s catalytic activity. Such hammerhead ribozyme constructs exhibit a turnover rate (kcat
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
) of about 1 molecule/minute and a Km
Enzyme kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction investigated...
on the order of 10 nanomolar.
The hammerhead ribozyme is arguably the best-characterized ribozyme. Its small size, thoroughly-investigated cleavage chemistry, known crystal structure, and its biological relevance make the hammerhead ribozyme particularly well-suited for biochemical and biophysical investigations into the fundamental nature of RNA catalysis.
Hammerhead ribozymes may play an important role as therapeutic agents; as enzymes which tailor defined RNA sequences, as biosensor
Biosensor
A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.It consists of 3 parts:* the sensitive biological element A biosensor is an analytical device for the detection of an analyte that combines a biological...
s, and for applications in functional genomics
Functional genomics
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
and gene discovery.
Species distribution
Hammerhead ribozymes are found in a wide range of plant viroids, helminths and more recently in a small number of archaebacteria and eubacteria:- Peach latent mosaic viroidPeach latent mosaic viroidPeach latent mosaic viroid is a type species from the genus Pelamoviroid, which belongs to the family Avsunviroidae. This family is characterized as having chloroplastic viroids with hammerhead ribozymes. Peach latent mosaic viroid is a 336-351nt circular RNA which has a branched formation. This...
- Eggplant latent viroid
- Avocado sunblotch viroidAvocado sunblotch viroidAvocado sunblotch viroid is an important disease affecting avocado trees.Infections result in lower yields and poorer quality fruit. ASBV is the smallest known viroid that infects plants and is transmitted by pollen and infected seeds or budwood....
- Velvet tobacco mottle virus Satellite RNA
- Schistosoma mansoniSchistosoma mansoniSchistosoma mansoni is a significant parasite of humans, a trematode that is one of the major agents of the disease schistosomiasis. The schistosomiasis caused by Schistosoma mansoni is intestinal schistosomiasis....
Satellite DNA - Dianthus caryophyllus viroid-like DNA
- Cherry small circular viroid-like RNA
- Newt
- Dolichopoda cave cricket
Chemistry of catalysis
The hammerhead ribozyme carries out a very simple chemical reaction that results in the breakage of the substrate strand of RNA, specifically at C17, the cleavage-site nucleotide. Although RNA cleavage is often referred to as hydrolysisHydrolysis
Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. It is the type of reaction that is used to break down certain polymers, especially those made by condensation polymerization...
, the mechanism employed does not in fact involve the addition of water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
. Rather, the cleavage reaction is simply an isomerization that consists of rearrangement of the linking phosphodiester bond. It is the same reaction, chemically, that occurs with random base
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...
-mediated RNA degradation, except that it is highly site-specific and the rate is accelerated 10,000-fold or more.
Cleavage by phosphodiester isomerization
The cleavage reaction is a phosphodiester isomerization reaction that is initiated by abstraction of the cleavage-site riboseRibose
Ribose is an organic compound with the formula C5H10O5; specifically, a monosaccharide with linear form H––4–H, which has all the hydroxyl groups on the same side in the Fischer projection....
2’-hydroxyl proton from the 2’-oxygen, which then becomes the attacking nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
in an “in-line” or SN2(P)-like reaction, although it is not known whether this proton is removed prior to or during the chemical step of the hammerhead cleavage reaction. (The cleavage reaction is technically not bimolecular, but behaves in the same way a genuine SN2(P) reaction does; it undergoes inversion of configuration subsequent to forming an associative transition-state
Transition state
The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
consisting of a pentacoordinated oxyphosphrane.) The attacking and leaving group
Leaving group
In chemistry, a leaving group is a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. Leaving groups can be anions or neutral molecules. Common anionic leaving groups are halides such as Cl−, Br−, and I−, and sulfonate esters, such as para-toluenesulfonate...
oxygens will both occupy the two axial positions in the trigonal bipyramidal transition-state structure as is required for an SN2-like reaction mechanism.
The 5’-product, as a result of this cleavage reaction mechanism, possesses a 2’,3’-cyclic phosphate terminus, and the 3’-product possesses a 5’-OH terminus, as with nonezymatic alkaline cleavage of RNA. The reaction is therefore, in principle, reversible, as the scissile phosphate remains a phosphodiester, and may thus act as a substrate for hammerhead RNA-mediated ligation without a requirement for ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
or a similar exogenous energy source. The hammerhead ribozyme-catalyzed reaction, unlike the formally identical non-enzymatic alkaline cleavage of RNA, is a highly sequence-specific cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate per molecule of enzyme per minute at pH 7.5 in 10 mM Mg2+ (so-called “standard reaction conditions” for the minimal hammerhead RNA sequence), depending upon the sequence of the particular hammerhead ribozyme construct measured. This represents an approximately 10,000-fold rate enhancement over the nonezymatic cleavage of RNA.
Requirement for divalent metal ions
All ribozymes were originally thought to be metallo-enzymesMetalloprotein
Metalloprotein is a generic term for a protein that contains a metal ion cofactor. Metalloproteins have many different functions in cells, such as enzymes, transport and storage proteins, and signal transduction proteins. Indeed, about one quarter to one third of all proteins require metals to...
, in the sense that they were assumed to require the presence of divalent cations, such as Mg2+
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
, for both folding and catalysis. It was presumed that hexahydrated magnesium
Magnesium
Magnesium is a chemical element with the symbol Mg, atomic number 12, and common oxidation number +2. It is an alkaline earth metal and the eighth most abundant element in the Earth's crust and ninth in the known universe as a whole...
ions, which exist in equilibrium with magnesium hydroxide
Magnesium hydroxide
Magnesium hydroxide is an inorganic compound with the chemical formula Mg2. As a suspension in water, it is often called milk of magnesia because of its milk-like appearance. The solid mineral form of magnesium hydroxide is known as brucite....
, could play the roles of general acid and general base, in a way analogous to those played by two histidines in RNase A. An additional role for divalent metal ions has also been proposed in the form of electrostatic stabilization of the transition-state
Transition state
The transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest energy along this reaction coordinate. At this point, assuming a perfectly irreversible reaction, colliding reactant molecules will always...
.
Not a metallo-enzyme
In 1998 it was discovered that the hammerhead ribozyme, as well as the VS ribozymeVS ribozyme
The Varkud satellite ribozyme is an RNA enzyme that carries out the cleavage of a phosphodiester bond.-Structure:The VS ribozyme is composed of 5 helices that form an H shape...
and hairpin ribozyme
Hairpin ribozyme
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 satellites of plant viruses...
, do not require the presence of metal ions for catalysis, provided a sufficiently high concentration of monovalent
Monovalent
Monovalent may refer to:*In chemistry, valence is a measure of the number of chemical bonds formed by the atoms of a given element. Monovalent is a synonym of univalent.*Monovalent ions contain one valence electron....
cation is present to permit the RNA to fold. This discovery suggested that the RNA itself, rather than serving as an inert, passive scaffold for the binding of chemically active divalent metal ions, is instead itself intimately involved in the chemistry of catalysis. The latest structural results, described below, indeed confirm that two invariant nucleotides, G12 and G8, are positioned consistent with roles as the general base and general acid in the hammerhead cleavage reaction.
Strictly speaking, therefore, the hammerhead ribozyme cannot be a metallo-enzyme.
Minimal ribozyme
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...
. The core region is flanked by Stems I, II and III, which are in general made of canonical Watson-Crick base-pairs
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...
but are otherwise not constrained with respect to sequence. The catalytic turnover rate of minimal hammerhead ribozymes is ~ 1/min (a range of 0.1/min to 10/min is commonly observed, depending upon the nonconserved sequences and the lengths of the three helical stems). Much of the experimental work carried out on hammerhead ribozymes has used a minimal construct.
Type I & type III hammerhead RNA
Structurally the hammerhead ribozyme is composed of three base paired helices, separated by short linkers of conserved sequences. These helices are called I, II and III. Hammerhead ribozymes can be classified into three types based on which helix the 5' and 3' ends are found in. If the 5' and 3' ends of the sequence contribute to stem I then it is a type I hammerhead ribozyme, and if the and 3' ends of the sequence contribute to stem III then it is a type III hammerhead ribozyme. Of the three possible topological types both type I and type III are common. Type II topology ribozymes have recently been discovered and are widely found in bacteria.Full-length ribozyme
The full-length hammerhead ribozyme consists of additional sequence elements in stems I and II that permit additional tertiary contacts to form. The tertiary interactions stabilize the active conformation of the ribozyme, resulting in cleavage rates up to 1000-fold greater than those for corresponding minimal hammerhead sequences.Minimal
Nature (journal)
Nature, first published on 4 November 1869, is ranked the world's most cited interdisciplinary scientific journal by the Science Edition of the 2010 Journal Citation Reports...
by Pley, Flaherty and McKay. Subsequently, an all-RNA minimal hammerhead ribozyme structure was published by Scott, Finch and Klug in Cell
Cell (journal)
Cell is a peer-reviewed scientific journal publishing research papers across a broad range of disciplines within the life sciences. Areas covered include molecular biology, cell biology, systems biology, stem cells, developmental biology, genetics and genomics, proteomics, cancer research,...
in early 1995.
The minimal hammerhead ribozyme is composed of three base paired helices, separated by short linkers of conserved sequence as shown in the crystal structure
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
. These helices are called I, II and III. The conserved uridine-turn links helix I to helix II and usually contains the sequence CUGA. Helix II and III are linked by a sequence GAAA. The cleavage reaction occurs between helix III and I, and is usually a C.
The structure of a full length ribozyme shows that there are extensive interactions between the loop of stem II and stem I.
Hammerhead ribozymes can be divided into three classes according to which of the three stems is formed from the 5' and 3' end of the sequence region. If stem III is formed from the 5' and 3' most parts of the sequence they are known as class III.
Structure-function
Despite the observations of hammerhead ribozyme catalysis in a crystal of the minimal hammerhead sequence in which the crystal lattice packing contacts by necessity confined the global positions of the distal termini of all three flanking helical stems, many biochemical experiments designed to probe transition-state interactions and the chemistry of catalysis appeared to be irreconcilable with the crystal structures.For example, the invariant core residues G5, G8, G12 and C3 in the minimal hammerhead ribozyme were each observed to be so fragile that changing even a single exocyclic functional group on any one of these nucleotides results in a dramatic reduction or abolition of catalytic activity, yet few of these appeared to form hydrogen bonds involving the Watson-Crick faces of these nucleotide bases in any of the minimal hammerhead structures, apart from a G-5 interaction in the product structure.
A particularly striking and only recently observed example consisted of G8 and G12, which were identified as possible participants in acid/base catalysis. Once it was demonstrated that the hammerhead RNA does not require divalent metal ions for catalysis, it gradually became apparent that the RNA itself, rather than passively bound divalent metal ions, must play a direct chemical role in any acid-base chemistry in the hammerhead ribozyme active site. It was however completely unclear how G12 and G8 could accomplish this, given the original structures of the minimal hammerhead ribozyme.
Other concerns included an NOE between U4 and U7 of the cleaved hammerhead ribozyme that had also been observed during NMR characterization, which suggested that these nucleotide bases must approach one another closer than about 6 Å, although close approach of U7 to U4 did not appear to be possible from the crystal structure. Finally, as previously discussed, the attacking nucleophile in the original structures, the 2’-OH of C17, was not in a position amenable to in-line attack upon the adjacent scissile phosphate.
Perhaps most worrisome were experiments that suggested the A-9 and scissile phosphates must come within about 4 Å of one another in the transition-state, based upon double phosphorothioate substitution and soft metal ion rescue experiments; the distance between these phosphates in the minimal hammerhead crystal structure was about 18 Å, with no clear mechanism for close approach if the Stem II and Stem I A-form helices were treated as rigid bodies. Taken together, these results appeared to suggest that a fairly large-scale conformational change must have take place in order to reach the transition-state within the minimal hammerhead ribozyme structure.
For these reasons, the two sets of experiments (biochemical vs. crystallographic) appeared not only to be at odds, but to be completely and hopelessly irreconcilable, generating a substantial amount of discord in the field. No compelling evidence for dismissing either set of experimental results was ever made successfully, although many claims to the contrary were made in favor of each.
Full-length
The crystal structure of the full-length hammerhead ribozyme thus clearly addresses all of the major concerns that appeared irreconcilable with the previous crystal structures of the minimal hammerhead ribozyme.
Structure and catalysis
The most likely explanation is then that G12, in the deprotonated form, is the general base, and the ribose of G8 is the general acid. The apparent kinetic pKa of the hammerhead ribozyme is 8.5, whereas the pKa of guanosine is about 9.5. It is possible that the pKa of G12 is perturbed from 9.5 to 8.5 in the hammerhead catalytic core; this hypothesis is currently the subject of intense investigation.
The close approach of the A9 and scissile phosphates requires the presence of a high concentration of positive charge. This is probably the source of the observation that divalent metal ions are required at low ionic strength, but can be dispensed with at higher concentrations of monovalent cations.
The reaction thus likely involves abstraction of the 2'-proton from C17, followed by nucleophilic attack upon the adjacent phosphate. As the bond between the scissile phosphorus and the 5'-O leaving group begins to break, a proton is supplied from the ribose of G8, which then likely reprotonates at the expense of a water molecule observed to hydrogen bond to it in the crystal structure.
Therapeutic applications
Modified hammerhead ribozymes are being tested as therapeutic agents. Synthetic RNAs containing sequences complementary to the mutant SOD1 mRNA and sequences necessary to form the hammerhead catalytic structure are being studied as a possible therapy for amyotrophic lateral sclerosisAmyotrophic lateral sclerosis
Amyotrophic lateral sclerosis , also referred to as Lou Gehrig's disease, is a form of motor neuron disease caused by the degeneration of upper and lower neurons, located in the ventral horn of the spinal cord and the cortical neurons that provide their efferent input...
. Work is also underway to find out whether they could be used to engineer HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
resistant lines of T-cells.
The therapeutic use of trans-cleaving hammerhead ribozymes has been severely hampered by its low-level activity in vivo. The true catalytic potential of trans-cleaving hammerhead ribozymes may be recouped in vivo and therapeutic derivatives are likely to complement other nucleic acid hybridizing therapeutic strategies. Already there are hammerhead ribozymes which are close to clinical application.