Hammerhead Ribozyme - Catalysis

Catalysis

In the natural state, a hammerhead RNA motif is a single strand of RNA, and although the cleavage takes place in the absence of enzymes, the hammerhead RNA itself is not a catalyst in its natural state, as it is consumed by the reaction and cannot catalyze multiple turnovers.

Trans-acting 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 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 (k_cat) of about 1 molecule/minute and a K_m 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.