Enzymes are complex proteins that, thanks to a series of different monomers, arranged in the right conformation, can perform all kinds of chemical reactions, at least, all the ones necessary for the sustaining of life.

Digging a little deeper into the chemical mechanisms of these proteins, always left with one question:

Why do just molecules made with these 20 amino acids, work as catalysts?

While working on another post, that explored the evolutionary origins of DNA, I became interested in ribozymes, and more in general, in the role and functions that RNA played in the origin of life.

Ribozymes are small filaments of RNA, sometimes in complex with proteins, that are able to catalyze a few reactions, one of the most common is self-cleavage. There are different hypotheses on the birth of the first genetic code, one of these points at RNA as a candidate, and claims that in particular conditions, it could work as the very first self-replicating entity by itself.

These multiple roles of RNA as nucleic acid, by encoding information in the form of nucleotides sequences, and as a catalyst, can give us hints on how, in the pre-biotic world, it could play a role in the evolution of these two functions, that later, have been replaced by more efficient and specialized molecules (DNA and proteins). While living space to these two inheritors, RNA has been forced to cover niche functions like transcription, translation, and regulation of genes, leaving us just a few pieces of evidence of its catalytic activity.

I’ve always seen RNA as the intermediate between DNA and proteins, not only because it is the molecule responsible for the transfer and translation of the information coded in the genome, to proteins, but also for its chemical properties.  Briefly, because RNA is found as a single filament, it can fold and create loops, and other complex structures, as proteins do, and, because the chemical bonds between its monomer are less stable in respect to the ones present in DNA, they can be broken and formed more easily, resulting in a more “dynamic” type of polymer.

Types of ribozymes

Ribozymes are catalytic RNA molecules, first identified in the early 1980s. They have the intrinsic ability to break and form covalent bonds in RNA molecules. In many ways, they can be compared to the protein enzymes which catalyze the cleavage of peptide bonds in other proteins or peptides. The majority of identified ribozymes belong to the small self-cleaving ribozyme family:

Hammerhead: first discovered in 1992 by observing the cycle of maturation of specific viroids. Constituted of 3 base-paired helixes of variable composition, connected by two single-stranded filaments. The latter is responsible for the catalytic activity of the complex and is highly preserved. The reaction is the cleavage of a phosphodiester bond to yield a 2‘,3’-cyclic  phosphate and a 5’-hydroxyl product, that could be performed both intra or inter molecularly. Divalent ions are necessary for its activity.

Hammerhead ribbon structure, colored by chain (PDB: 379D) 

Twister ribozyme: representatives of the twister ribozyme class are found in all domains of life, so-called because their conserved secondary structure resembles the ancient Egyptian hieroglyph “twisted flax.” Its rate of self-cleavage is slightly higher in respect to hammerhead.

Twister ribozyme ribbon structure, colored by chain (PDB: 4QJH)

HDV: this stretch of 85 nucleotides is responsible for the cleavage and maturation of small mRNAs necessary for the lifecycle of a small virus-like particle [∼1,700-nucleotide (nt)]. The pathogen is responsible for hepatitis D, which infects only patients who have hepatitis B. The active site of this ribozyme is formed by a double pseudoknot structure.

HDV ribozyme ribbon structure, colored by chain (PDB: 4PRF)


Molecular mechanism of self-cleavage

To discover new members of the ribozyme family, often bioinformatic analyses are carried out on databases with a big collection of genomic sequences. Screening in vitro some of these sequences by looking for common motives, found in already discovered ribozymes, allows us to discover new members. This was the case with Pistol ribozyme.

The pistol ribozyme is made of 2 different RNA filaments twisted together in forming the active complex. The bigger filament ( circa50 nt) is responsible for the catalytic activity, the smaller one acts as the substrate of the reaction, and will be cut in a specific region: between G53 and U54 (aka G10 and U11, depending on the enumeration method). There are variants in nature, but 10 residues are highly preserved, showing that they are responsible for the mechanism of reaction or the stability of the complex.

Secondary structure of the env25 pistol ribozyme showing highly conserved residues in red
(Ren A et al, 2016) 

The reaction is a 2′-O-transphosphorylation, it involves the breaking of the phosphodiester bond between the two nucleotides that act as substrate, resulting in a cyclic phosphate between 3’ and 5’ oxygen atoms, and a new hydroxyl group in the 5’ position of the other nucleotide. 

  • α: The correct alignment between the 2’O atom (nucleophile), the P (electrophile), and the 5’O that act as leaving group.
  • β: the neutralization of the negative charge present in the non-bridging O atom of the phosphate group.
  • γ:  The deprotonation of the 2’ O by a base; this is necessary for the activation of the nucleophile species, that, then will “attack” the electrophile.
  • δ:  stabilization of the 5’O as leaving group

Schematic representation of the possible factors explaining the catalytic activity of self-cleaving ribozymes (Serrano-Aparicio et al, 2021)

These strategies work together to achieve the final reaction, but the way in which they contribute to the mechanism of a particular ribozyme or enzyme changes depending on different variants, conditions, and residue availability.

In the case of pistol ribozyme, the suggested mechanism is similar to the one identified also in hammerhead. A crucial role in both, the activation of the 2’ O and the stabilization of the 5’O leaving group (δ), is mediated by Mg2+ ions, able to coordinate different residues in the active site.

The acid-base catalysis is performed by 2 residues in the active site: G40 and G32 that act as a base (γ) and acid (β) respectively, promoting the transfer of H and electrons in the reaction site.


In these studies, classic molecular dynamics, and hybrid QM/MM (quantum mechanics / molecular mechanics) models are used to evaluate the probability and characteristics of each proposed mechanism. These analyses are used to evaluate some properties like the possible angles between residues, the stable conformation in solution, the effect of solvent and ions in the reaction, the energy barriers, and so on. The starting point often is a crystallographic model, that needs to be modified, to better represent the active complex ( free in solution).

The models are not perfect and require some parametrization of initial conditions, and a trade-off between accuracy and computational power, but have a good level of trustfulness and predictivity.






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