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 (ribozymes) 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 (i.e. performs self-cleavage) 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 (mostly invariant) 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 (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 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 (SINEs) 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 (the so-called HH9 and HH10) 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
… excerpt ends here. Continue reading the full article.
