Hammerhead ribozyme
The hammerhead ribozyme is an RNA motif that catalyzes reversible cleavage and ligation reactions at a specific site within an RNA molecule. It is one of several catalytic RNAs known to occur in nature. 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. Named for the resemblance of early secondary structure diagrams to a hammerhead shark, hammerhead ribozymes were originally discovered in two classes of plant virus-like RNAs: satellite RNAs and viroids. They are also known in some classes of retrotransposons, including the retrozymes. The hammerhead ribozyme motif has been ubiquitously reported in lineages across the tree of life.
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.
Catalysis
In its natural state, a hammerhead RNA motif is a single strand of RNA. Although the cleavage takes place in the absence of protein enzymes, the hammerhead RNA itself is not a catalyst in its natural state, as it is consumed by the reaction and therefore cannot catalyze multiple turnovers.Trans-acting hammerhead constructs can be engineered such that they consist of two interacting RNA strands, with one strand composing a hammerhead ribozyme that cleaves the other strand. 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 trans-acting hammerhead ribozyme sequence that is catalytically active consists of three base-paired stems flanking a central core of 15 conserved nucleotides, as shown. The conserved central bases, with few exceptions, are essential for ribozyme's catalytic activity. Such hammerhead ribozyme constructs exhibit in vitro a turnover rate 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 biosensors, and for applications in functional genomics and gene discovery.
Species distribution
In 1986, the first hammerhead ribozymes were found in RNA plant pathogens like viroids and viral satellites. One year later, a hammerhead ribozyme was also reported in the satellite DNA of newt genomes. New examples of this ribozyme were then found in the genomes of unrelated organisms like schistosomes, cave crickets, Arabidopsis thaliana and a few mammals like rodents and the platypus. In 2010, it was found that the hammerhead ribozyme occurs in a wide variety of bacterial and eukaryotic genomes, including in humans. Similar reports confirmed and extended these observations, unveiling the hammerhead ribozyme as a ubiquitous catalytic RNA in all life kingdoms.Most eukaryotic hammerhead ribozymes are related to a kind of short interspersed retroelements called retrozymes, which express as small circular RNAs. However, an exceptional group of strikingly conserved hammerheads can be found in the genomes of all amniotes. These hammerhead ribozymes occur in the introns of a few specific genes and point to a preserved biological role during pre-mRNA biosynthesis. In 2021, novel Hepatitis D virus genomes of circular RNA from diverse animals were found to encode hammerhead ribozymes similar to those present in plant viroids and viral satellites. A massive bioinformatic search of deltavirus-like agents around the globe has uncovered hundreds of examples of circular RNA genomes carrying hammerhead motifs, indicating that not only this ribozyme but small circular RNAs with ribozymes are ubiquitous molecules in the biosphere.
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 hydrolysis, 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.Cleavage by phosphodiester isomerization
The cleavage reaction is a phosphodiester isomerization reaction that is initiated by abstraction of the cleavage-site ribose 2'-hydroxyl proton from the 2'-oxygen, which then becomes the attacking nucleophile in an "in-line" or SN2-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 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 nonenzymatic alkaline cleavage of RNA. The reaction is therefore 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+, depending upon the sequence of the particular hammerhead ribozyme construct measured. This represents an approximately 10,000-fold rate enhancement over the nonenzymatic cleavage of RNA.
Requirement for divalent metal ions
All ribozymes were originally thought to be metallo-enzymes. It was assumed that divalent metal ions like Mg2+ were thought to have two roles: Promote proper folding of RNA and to form the catalytic core. Since RNA itself did not contain enough variation in the functional groups, metal ions were thought to play a role at the active site, as was known about proteins. The proposed mechanism for the Mg2+ ion was: the deprotonation of the 2'-OH group by a Magnesium.aqua.hydroxy complex bound by the pro-R oxygen at the phosphate-cleavage site, followed by nucleophilic attack of the resultant 2'-alkaoxide on the scissile phosphate forming a pentacoordinate phosphate intermediate. The last step is the departing of the 5' leaving group, yielding a 2',3'-cyclic phosphate with an inverted configuration.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.
Not a metallo-enzyme
In 1998 it was discovered that the hammerhead ribozyme, as well as the VS ribozyme 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.