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Ribozyme
Ribozyme
Ribozyme
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3D structure of a hammerhead ribozyme

Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like proteins), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems.[1]

The most common activities of natural or in vitro evolved ribozymes are the cleavage or ligation of RNA and DNA, and peptide bond formation.[2] For example, the smallest ribozyme known (GUGGC-3') can aminoacylate a GCCU-3' sequence in the presence of Phenylalanyl-Adenosine Monophosphate.[3] Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, leadzyme, and the hairpin ribozyme.

Researchers who are investigating the origins of life through the RNA world hypothesis have been working on discovering a ribozyme with the capacity to self-replicate, which would require it to have the ability to catalytically synthesize polymers of RNA. This should be able to happen in prebiotically plausible conditions with high rates of copying accuracy to prevent degradation of information, but also allowing for the occurrence of occasional errors during the copying process to allow for Darwinian evolution to proceed.[4]

Attempts have been made to develop ribozymes as therapeutic agents, as enzymes which target defined RNA sequences for cleavage, as biosensors, and for applications in functional genomics and gene discovery.[5]

Discovery

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Schematic showing ribozyme cleavage of RNA

Before the discovery of ribozymes, enzymes—which were defined [solely] as catalytic proteins—were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures.[6] These ribozymes were found in the intron of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman shared the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA".[7] The term ribozyme was first introduced by Kelly Kruger et al. in a paper published in Cell in 1982.[1]

It had been a firmly established belief in biology that catalysis was reserved for proteins. However, the idea of RNA catalysis is motivated in part by the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? The concept of "ribonucleic acids as catalysts" circumvents this problem. RNA, in essence, can be both the chicken and the egg.[8]

In the 1980s, Thomas Cech, at the University of Colorado Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for the splicing reaction, he found that the intron could be spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University, was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year[which?], Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component.

Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin – or hammerhead – shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme were made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

Structure and mechanism

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Despite having only four choices for each monomer unit (nucleotides), compared to 20 amino acid side chains found in proteins, ribozymes have diverse structures and mechanisms. In many cases they are able to mimic the mechanism used by their protein counterparts. For example, in self cleaving ribozyme RNAs, an in-line SN2 reaction is carried out using the 2' hydroxyl group as a nucleophile attacking the bridging phosphate and causing 5' oxygen of the N+1 base to act as a leaving group. In comparison, RNase A, a protein that catalyzes the same reaction, uses a coordinating histidine and lysine to act as a base to attack the phosphate backbone.[2][clarification needed]

Like many protein enzymes, metal binding is also critical to the function of many ribozymes.[9] Often these interactions use both the phosphate backbone and the base of the nucleotide, causing drastic conformational changes.[10] There are two mechanism classes for the cleavage of a phosphodiester backbone in the presence of metal. In the first mechanism, the internal 2'- OH group attacks the phosphorus center in a SN2 mechanism. Metal ions promote this reaction by first coordinating the phosphate oxygen and later stabling the oxyanion. The second mechanism also follows a SN2 displacement, but the nucleophile comes from water or exogenous hydroxyl groups rather than RNA itself. The smallest ribozyme is UUU, which can promote the cleavage between G and A of the GAAA tetranucleotide via the first mechanism in the presence of Mn2+. The reason why this trinucleotide (rather than the complementary tetramer) catalyzes this reaction may be because the UUU-AAA pairing is the weakest and most flexible trinucleotide among the 64 conformations, which provides the binding site for Mn2+.[11]

Phosphoryl transfer can also be catalyzed without metal ions. For example, pancreatic ribonuclease A and hepatitis delta virus (HDV) ribozymes can catalyze the cleavage of RNA backbone through acid-base catalysis without metal ions.[12][13] Hairpin ribozyme can also catalyze the self-cleavage of RNA without metal ions, but the mechanism for this is still unclear.[13]

Ribozyme can also catalyze the formation of peptide bond between adjacent amino acids by lowering the activation entropy.[12]

Ribozyme structure pictures
Image showing the diversity of ribozyme structures. From left to right: leadzyme, hammerhead ribozyme, twister ribozyme

Activities

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A ribosome is a biological machine that utilizes a ribozyme to translate RNA into proteins.

Although ribozymes are quite rare in most cells, their roles are sometimes essential to life. For example, the functional part of the ribosome, the biological machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors.[14] In a model system, there is no requirement for divalent cations in a five-nucleotide RNA catalyzing trans-phenylalanation of a four-nucleotide substrate with 3 base pairs complementary with the catalyst, where the catalyst/substrate were devised by truncation of the C3 ribozyme.[15]

The best-studied ribozymes are probably those that cut themselves or other RNAs, as in the original discovery by Cech[16] and Altman.[17] However, ribozymes can be designed to catalyze a range of reactions[18] or they have them naturally. Some of these activities include the following:[19]

RNA may catalyze folding of the pathological protein conformation of a prion in a manner similar to that of a chaperonin.[20]

Ribozymes and the origin of life

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RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule rather than dividing these functions between DNA and protein as they are today; this hypothesis is known as the "RNA world hypothesis" of the origin of life.[21] Since nucleotides and RNA (and thus ribozymes) can arise by inorganic chemicals, they are candidates for the first enzymes, and in fact, the first "replicators" (i.e., information-containing macro-molecules that replicate themselves). An example of a self-replicating ribozyme that ligates two substrates to generate an exact copy of itself was described in 2002.[22] The discovery of the catalytic activity of RNA solved the "chicken and egg" paradox of the origin of life, solving the problem of origin of peptide and nucleic acid central dogma. According to this scenario, at the origin of life, all enzymatic activity and genetic information encoding was done by one molecule: RNA.

Ribozymes have been produced in the laboratory that are capable of catalyzing the synthesis of other RNA molecules from activated monomers under very specific conditions, these molecules being known as RNA polymerase ribozymes.[23] The first RNA polymerase ribozyme was reported in 1996, and was capable of synthesizing RNA polymers up to 6 nucleotides in length.[24] Mutagenesis and selection has been performed on an RNA ligase ribozyme from a large pool of random RNA sequences,[25] resulting in isolation of the improved "Round-18" polymerase ribozyme in 2001 which could catalyze RNA polymers now up to 14 nucleotides in length.[26] Upon application of further selection on the Round-18 ribozyme, the B6.61 ribozyme was generated and was able to add up to 20 nucleotides to a primer template in 24 hours, until it decomposes by cleavage of its phosphodiester bonds.[27]

The rate at which ribozymes can polymerize an RNA sequence multiples substantially when it takes place within a micelle.[28]

The next ribozyme discovered was the "tC19Z" ribozyme, which can add up to 95 nucleotides with a fidelity of 0.0083 mutations/nucleotide.[29] Next, the "tC9Y" ribozyme was discovered by researchers and was further able to synthesize RNA strands up to 206 nucleotides long in the eutectic phase conditions at below-zero temperature,[30] conditions previously shown to promote ribozyme polymerase activity.[31]

The RNA polymerase ribozyme (RPR) called tC9-4M was able to polymerize RNA chains longer than itself (i.e. longer than 177 nt) in magnesium ion concentrations close to physiological levels, whereas earlier RPRs required prebiotically implausible concentrations of up to 200 mM. The only factor required for it to achieve this was the presence of a very simple amino acid polymer called lysine decapeptide.[32]

The most complex RPR synthesized by that point was called 24-3, which was newly capable of polymerizing the sequences of a substantial variety of nucleotide sequences and navigating through complex secondary structures of RNA substrates inaccessible to previous ribozymes. In fact, this experiment was the first to use a ribozyme to synthesize a tRNA molecule.[33] Starting with the 24-3 ribozyme, Tjhung et al.[34] applied another fourteen rounds of selection to obtain an RNA polymerase ribozyme by in vitro evolution termed '38-6' that has an unprecedented level of activity in copying complex RNA molecules. However, this ribozyme is unable to copy itself and its RNA products have a high mutation rate. In a subsequent study, the researchers began with the 38-6 ribozyme and applied another 14 rounds of selection to generate the '52-2' ribozyme, which compared to 38-6, was again many times more active and could begin generating detectable and functional levels of the class I ligase, although it was still limited in its fidelity and functionality in comparison to copying of the same template by proteins such as the T7 RNA polymerase.[35]

An RPR called t5(+1) adds triplet nucleotides at a time instead of just one nucleotide at a time. This heterodimeric RPR can navigate secondary structures inaccessible to 24-3, including hairpins. In the initial pool of RNA variants derived only from a previously synthesized RPR known as the Z RPR, two sequences separately emerged and evolved to be mutualistically dependent on each other. The Type 1 RNA evolved to be catalytically inactive, but complexing with the Type 5 RNA boosted its polymerization ability and enabled intermolecular interactions with the RNA template substrate obviating the need to tether the template directly to the RNA sequence of the RPR, which was a limitation of earlier studies. Not only did t5(+1) not need tethering to the template, but a primer was not needed either as t5(+1) had the ability to polymerize a template in both 3' → 5' and 5' 3 → 3' directions.[36]

A highly evolved[vague] RNA polymerase ribozyme was able to function as a reverse transcriptase, that is, it can synthesize a DNA copy using an RNA template.[37] Such an activity is considered[by whom?] to have been crucial for the transition from RNA to DNA genomes during the early history of life on earth. Reverse transcription capability could have arisen as a secondary function of an early RNA-dependent RNA polymerase ribozyme.

An RNA sequence that folds into a ribozyme is capable of invading duplexed RNA, rearranging into an open holopolymerase complex, and then searching for a specific RNA promoter sequence, and upon recognition rearrange again into a processive form that polymerizes a complementary strand of the sequence. This ribozyme is capable of extending duplexed RNA by up to 107 nucleotides, and does so without needing to tether the sequence being polymerized.[38]

A short 20-nucleotide RNA variant ribozyme was identified that self-reproduces via template directed ligation of two 10 nucleotide oligomers.[39] This minimal kind of RNA self-reproduction was discovered in a random pool of oligmers, and may represent an early step in the emergence of an RNA based genetic system from primordial components.[39]

Ribozyme based origin of sexual reproduction

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Sexual reproduction might have been present in the RNA world that preceded DNA cellular life forms.[40] Early cellular life forms having genomes with single copies of essential RNA ribozyme molecules would likely have been vulnerable to environmental damaging conditions that could block replication of an essential ribozyme thus causing cell death. Merger of two such damaged early cells (sexual interaction) would allow undamaged combinations of RNA segments to come together, thus facilitating formation of a functional genome and allowing survival of the cell and ability to reproduce.

Artificial ribozymes

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Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificially produced self-cleaving RNAs with good enzymatic activity have been produced. Tang and Breaker[41] isolated self-cleaving RNAs by in vitro selection of RNAs originating from random-sequence RNAs. Some of the synthetic ribozymes that were produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme.

In 2015, researchers at Northwestern University and the University of Illinois Chicago engineered a tethered ribosome that works nearly as well as the authentic cellular component that produces all the proteins and enzymes within the cell. Called Ribosome-T, or Ribo-T, the artificial ribosome was created by Michael Jewett and Alexander Mankin.[42] The techniques used to create artificial ribozymes involve directed evolution. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcribing them with reverse transcriptase into various cDNA and amplified with error-prone PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are covalently linked to the substrate. If a molecule possesses the desired ligase activity, a streptavidin matrix can be used to recover the active molecules.

Lincoln and Joyce used in vitro evolution to develop ribozyme ligases capable of self-replication in about an hour, via the joining of pre-synthesized highly complementary oligonucleotides.[43]

Although not true catalysts, the creation of artificial self-cleaving riboswitches, termed aptazymes, has also been an active area of research. Riboswitches are regulatory RNA motifs that change their structure in response to a small molecule ligand to regulate translation. While there are many known natural riboswitches that bind a wide array of metabolites and other small organic molecules, only one ribozyme based on a riboswitch has been described: glmS.[44] Early work in characterizing self-cleaving riboswitches was focused on using theophylline as the ligand. In these studies, an RNA hairpin is formed which blocks the ribosome binding site, thus inhibiting translation. In the presence of the ligand, in these cases theophylline, the regulatory RNA region is cleaved off, allowing the ribosome to bind and translate the target gene. Much of this RNA engineering work was based on rational design and previously determined RNA structures rather than directed evolution as in the above examples. More recent work has broadened the ligands used in ribozyme riboswitches to include thymine pyrophosphate. Fluorescence-activated cell sorting has also been used to engineering aptazymes.[45]

Applications

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Ribozymes have been proposed and developed for the treatment of disease through gene therapy. One major challenge of using RNA-based enzymes as a therapeutic is the short half-life of the catalytic RNA molecules in the body. To combat this, the 2' position on the ribose is modified to improve RNA stability. One area of ribozyme gene therapy has been the inhibition of RNA-based viruses.

A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection.[46][47]

Similarly, ribozymes have been designed to target the hepatitis C virus RNA, SARS coronavirus (SARS-CoV),[48] Adenovirus[48] and influenza A and B virus RNA.[49][50][51][48] The ribozyme is able to cleave the conserved regions of the virus's genome, which has been shown to reduce the virus in mammalian cell culture.[52] Despite these efforts by researchers, these projects have remained in the preclinical stage.

Known ribozymes

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Well-validated naturally occurring ribozyme classes:

See also

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Notes and references

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Further reading

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Ribozyme

View on Grokipedia
from Grokipedia
A ribozyme is a ribonucleic acid () molecule or complex of RNA molecules that functions as a biological catalyst, accelerating specific chemical reactions in a manner analogous to protein-based enzymes. These catalytic RNAs were first identified in the early 1980s, fundamentally altering the by revealing that RNA could serve dual roles as both genetic information carrier and active biochemical agent. The breakthrough came independently from two researchers: In 1982, Thomas R. Cech demonstrated that the intervening sequence () in the ribosomal precursor of the ciliate protozoan Tetrahymena thermophila undergoes self-splicing, excising itself and ligating the flanking exons without requiring protein enzymes, thus establishing the first example of an RNA catalyst. Shortly thereafter, in 1983, showed that the RNA subunit of ribonuclease P (RNase P), an enzyme essential for tRNA maturation in cells including Escherichia coli, performs the catalytic cleavage of precursor tRNAs independently of its protein components. For these discoveries, Cech and Altman were jointly awarded the 1989 , recognizing the paradigm-shifting insight that RNA possesses enzymatic properties. Ribozymes encompass a diverse array of structures and functions, ranging from large, intricate molecules to small, compact motifs. Notable examples include the self-splicing found in organelles and , which catalyze ; the RNase P ribozyme, which processes tRNA precursors; and smaller self-cleaving ribozymes such as the hammerhead, discovered in 1986 within plant satellite RNAs like that of ringspot virus, and the delta (HDV) ribozyme, identified in 1988, both of which facilitate precise cleavage. Additionally, the peptidyl transferase center of the , composed of (rRNA), acts as a ribozyme to catalyze formation during protein synthesis, underscoring RNA's central role in translation. The existence of ribozymes provides strong evidence for the RNA world hypothesis, positing that RNA preceded DNA and proteins in early life evolution, serving as both genome and catalyst before the emergence of more efficient protein enzymes. Beyond fundamental biology, ribozymes have inspired biotechnological applications, including RNA-based therapeutics for (e.g., via ribozyme-mediated cleavage of disease-causing RNAs) and tools for , such as in vitro-evolved ribozymes that perform ligation, polymerization, or metabolite sensing. Ongoing research continues to uncover novel ribozymes through bioinformatics and experimental selection, expanding our understanding of RNA's catalytic potential in diverse organisms and contexts.

Fundamentals and History

Definition and Properties

A ribozyme is a molecule, or a molecule containing an RNA moiety, that functions as a biological by accelerating specific chemical reactions. Like protein enzymes, ribozymes lower the of these reactions without being altered or consumed in the process, thereby enabling efficient biochemical transformations in cells. Ribozymes exhibit several distinctive properties that underpin their catalytic capabilities. They typically bind substrates through sequence-specific base-pairing interactions, which provide in recognition akin to Watson-Crick hybridization. The is formed by a conserved catalytic core comprising specific motifs and secondary structures that position reactive groups for . Many ribozymes, particularly self-cleaving ones, demonstrate the ability to act on themselves, facilitating intramolecular reactions. Additionally, their activity often depends on divalent metal ions, such as Mg²⁺, which neutralize the negative charge of the phosphate backbone to promote proper folding and directly participate in the by coordinating substrates or activating nucleophiles. In kinetic terms, ribozymes share fundamental similarities with protein enzymes, displaying Michaelis-Menten behavior characterized by parameters such as the (kcat) and the Michaelis constant (Km), which reflect substrate affinity and catalytic efficiency. For instance, self-cleaving ribozymes can achieve rate enhancements of 103 to 106-fold over uncatalyzed cleavage rates, though this is generally less than the 1010 to 1015-fold accelerations typical of protein counterparts for analogous reactions. Thermodynamically, ribozyme function relies on the RNA folding into precise secondary (e.g., helices and loops) and tertiary structures to form the active conformation, a process stabilized by ionic interactions that screen electrostatic repulsions; optimal folding and activity are thus highly sensitive to salt concentration and cation type. First identified in the early , these properties highlight ribozymes' role as versatile catalysts in biological systems.

Discovery and Early Research

The discovery of ribozymes began in 1982 when and his colleagues at the identified self-splicing introns in the precursor of the ciliate protozoan Tetrahymena thermophila. In their key experiments, Cech's team conducted transcription and splicing assays using purified pre-rRNA transcripts, demonstrating that the intervening sequence (IVS) RNA could excise itself and cyclize without requiring protein enzymes, indicating RNA . This finding was initially surprising, as the team had anticipated protein involvement in the splicing process, but studies revealed the RNA component alone was sufficient for the reaction under physiological conditions like monovalent and divalent cations. In 1983, and collaborators at reported the catalytic role of the subunit in ribonuclease P (RNase P), an enzyme essential for tRNA maturation in . Through fractionation and reconstitution experiments with Escherichia coli extracts, they isolated the RNA moiety (M1 RNA) and showed it could cleave precursor tRNA substrates in vitro when provided with magnesium ions, independent of the protein subunit, thus establishing it as the catalytic component. Altman's prior work in the had already suggested RNase P contained an component, but these 1983 assays confirmed its enzymatic activity, paralleling Cech's observations. The groundbreaking revelations by Cech and Altman culminated in the 1989 , awarded jointly for their discovery of catalytic properties in . This recognition highlighted how ribozymes expanded the understanding of RNA's functional versatility, shifting the paradigm from viewing RNA solely as a passive messenger in the to recognizing it as an active biocatalyst capable of both storing genetic information and accelerating chemical reactions. Early research thus laid the foundation for exploring RNA's dual roles, influencing subsequent studies on its evolutionary and biochemical significance.

Biophysical Foundations

Structural Features

Ribozymes exhibit characteristic secondary structures primarily formed through Watson-Crick base pairing, resulting in double-helical stems, internal and terminal loops, and unpaired bulges that contribute to overall folding and stability. These elements create a scaffold where stems provide rigidity via A-form helices, while loops and bulges introduce flexibility and sites for tertiary interactions. For instance, in small self-cleaving ribozymes, secondary structures often consist of three helical domains connected at a central , as seen in the hammerhead ribozyme. Tertiary structures of ribozymes are stabilized by motifs that pack helices and single-stranded regions into compact architectures, including pseudoknots, coaxial helical stacking, and A-minor motifs. Pseudoknots arise when a single-stranded loop base-pairs with a distant complementary sequence, forming an additional helix that interlocks with existing stems, as observed in variants of the hammerhead ribozyme (PDB: 8YDC). Coaxial stacking aligns adjacent helices end-to-end for extended rigid domains, while A-minor interactions involve adenine bases inserting into the minor grooves of helices to mediate long-range contacts, exemplified in the P4-P6 domain of group I introns (PDB: 1GID). Crystal structures, such as that of the hatchet ribozyme (PDB: 6JQ6), reveal pseudosymmetric dimeric scaffolds with these motifs enabling tight packing essential for function. Divalent ions, particularly Mg²⁺, play a critical role in ribozyme tertiary folding and formation through coordination to phosphate backbones and nucleobases. In many ribozymes, Mg²⁺ ions occupy positions in the , often in a two-metal-ion mechanism where they neutralize negative charges and position substrates for catalysis, as detailed in structures of the hammerhead and group I introns. Environmental factors like and temperature influence stability; optimal folding typically occurs at neutral (around 7-8) and moderate temperatures (e.g., 37-50°C), where deviations can disrupt base pairing or ion binding, leading to misfolding. For example, elevated temperatures denature small ribozymes, while low protonates key residues, impairing Mg²⁺ coordination. Structural diversity among ribozymes reflects their functional specialization, with small ribozymes (e.g., hammerhead, ~50 nucleotides) adopting compact, single-domain folds dominated by local interactions, whereas large ribozymes like group I introns (~400 nucleotides) feature modular architectures with multiple domains assembled via peripheral elements. This modularity in group I introns allows hierarchical folding, starting from conserved core helices and progressing to docking of peripheral domains (PDB: 1U6B). In contrast, the compact nature of small ribozymes enables rapid folding but limits complexity compared to the expansive, multi-subunit-like organization of larger ones.

Catalytic Mechanisms

Ribozymes accelerate chemical reactions through several general strategies that mimic enzymatic , primarily involving the manipulation of group transfers. These mechanisms rely on the RNA backbone and nucleobases to facilitate nucleophilic attacks and stabilize transition states, often in conjunction with divalent metal ions. General acid-base is achieved via nucleobases such as or , which donate or accept protons to activate s or stabilize leaving groups; for instance, the N1 of can act as a general acid by protonating the departing 5'-oxygen during cleavage. Metal ion-mediated Lewis acid further enhances reactivity, where ions like Mg²⁺ coordinate to non-bridging oxygens, neutralizing negative charges and polarizing the center to promote bond breakage. Substrate positioning is another critical strategy, wherein RNA's tertiary structure orients reactive groups into optimal geometries, such as aligning a 2'-hydroxyl for an inline attack on the adjacent atom. The kinetics of ribozyme catalysis generally follow the Michaelis-Menten model, describing the rate of reaction as dependent on enzyme (ribozyme) and substrate concentrations: v=kcat[E][S]Km+[S]v = \frac{k_{\text{cat}} [E][S]}{K_m + [S]} where vv is the initial velocity, kcatk_{\text{cat}} is the turnover number, [E][E] and [S][S] are the concentrations of ribozyme and substrate, and KmK_m reflects substrate affinity. For natural ribozymes, typical kcatk_{\text{cat}} values range from 1 to 100 min⁻¹, indicating moderate catalytic efficiency compared to uncatalyzed rates, with enhancements up to 10¹⁰-fold for phosphodiester cleavage. In activation mechanisms, such as phosphodiester bond cleavage, the 2'-OH group acts as a nucleophile in an S_N2-like inline attack on the phosphorus, forming a trigonal bipyramidal transition state that leads to a 2',3'-cyclic phosphate intermediate and a 5'-OH leaving group. Transition state stabilization occurs through electrostatic interactions, including hydrogen bonding from nearby nucleobases or metal ions that delocalize negative charge on the oxygens. Despite these capabilities, ribozyme exhibits limitations inherent to 's chemical composition, resulting in slower rates than protein enzymes. possesses fewer diverse functional groups—primarily the 2'-OH, nucleobase nitrogens, and oxygens—limiting its ability to form extensive networks or hydrophobic pockets for precise substrate discrimination and binding. This constraint often necessitates reliance on metal ions for charge neutralization, and overall rate enhancements rarely exceed 10⁷-fold for small-molecule reactions, far below the 10¹²- to 10¹⁷-fold typical of proteins. loops and helices provide the structural motifs that support these mechanisms by organizing catalytic residues.

Natural Roles

Biological Activities

Natural ribozymes catalyze a diverse array of chemical reactions critical to cellular , with the predominant activities centered on cleavage and ligation, as well as formation. Cleavage reactions typically involve site-specific of RNA backbones, enabling precise RNA fragmentation, while ligation facilitates the joining of RNA segments through formation. formation, a distinct non-phosphoryl transfer activity, underpins protein synthesis by linking . These reaction types highlight the versatility of in mediating key biochemical transformations without protein involvement. In cellular contexts, ribozyme activities are integral to RNA processing, where cleavage and ligation support splicing and maturation to produce functional transcripts. During , the peptidyl transferase activity drives the elongation of polypeptide chains within the . In viroid replication, self-cleavage processes multimeric RNA forms generated by rolling-circle mechanisms, ensuring the production of mature infectious units. These roles underscore ribozymes' contributions to , , and pathogen propagation.90170-5) The physiological efficiency of natural ribozymes in vivo frequently relies on cofactors such as GTP, which energizes certain ligation steps, or proteins that stabilize structures and enhance turnover rates. Many ribozymes exhibit multi-turnover kinetics, processing multiple substrates sequentially, as seen in tRNA maturation pathways, whereas others perform single-turnover reactions, such as intron self-excision, limiting them to one catalytic event per molecule. This cofactor dependence and kinetic variability optimize ribozyme function within complex cellular environments. Ribozymes demonstrate remarkable evolutionary conservation, occurring ubiquitously across all domains of life—including , , and eukaryotes—which points to their primordial emergence and enduring functional importance.

Examples of Natural Ribozymes

Natural ribozymes encompass a diverse array of catalytic RNAs found across all domains of life, performing essential roles in RNA processing and regulation. Small self-cleaving ribozymes represent one major class, typically comprising compact structures of 50–200 that catalyze site-specific cleavage to facilitate RNA maturation or replication. These include the hammerhead ribozyme, first identified in plant viroids and satellite RNAs, which adopts a three-way helical junction structure and enables the processing of multimeric transcripts during rolling-circle replication in eukaryotes such as , amphibians, and schistosomes. The hairpin ribozyme, occurring in satellite RNAs of plant viruses like ringspot virus, features a complex secondary structure with four helical domains and two internal loops, supporting self-cleavage to generate unit-length RNAs for viral propagation. Similarly, the VS ribozyme from the Varkud satellite RNA in mitochondria exhibits a multi-domain with six helical segments, where it cleaves to resolve RNA dimers during mitochondrial RNA maintenance. The glmS ribozyme, linked to a in like , forms a double structure and is activated by glucosamine-6-phosphate to cleave the mRNA, thereby autoregulating the synthesis of this metabolite. Larger splicing ribozymes, often exceeding 200 , mediate intron removal in precursor RNAs through more intricate folding patterns. Group I introns, exemplified by the Tetrahymena thermophila pre-rRNA , possess a conserved core with 10 helical domains and an internal guide sequence, enabling self-splicing that excises the and ligates exons in organellar genes across fungi, protists, , , and phages. Group II introns, prevalent in bacterial and organellar genomes such as those of mitochondria, feature six domains including a lariat-forming , and catalyze self-splicing to process pre-mRNAs, tRNAs, and rRNAs while also exhibiting mobility as retroelements. RNase P, a ubiquitous ribonucleoprotein complex, contains a catalytic RNA subunit (350–500 in eukaryotes) with helical elements like P4 and P10–P12 that processes the 5' leader sequence from tRNA precursors, essential for tRNA maturation in all prokaryotes and eukaryotes. The ribosomal peptidyl transferase center, embedded within the 23S rRNA of the large ribosomal subunit, constitutes a universal ribozyme that catalyzes formation during protein synthesis across all life forms. This A-site region, formed by conserved rRNA helices such as H80 and H89, positions aminoacyl- and peptidyl-tRNAs to drive the core transpeptidation reaction, underscoring RNA's ancient catalytic primacy . Additional classes of natural ribozymes include the HDV ribozyme from hepatitis delta virus, a compact 85-nucleotide motif with a double that undergoes self-cleavage to process viral antigenomic RNAs during replication in hepatocytes. More recently discovered via bioinformatics, the twister ribozyme features a four-stem structure in diverse , , and eukaryotes, where it performs self-cleavage potentially to regulate mRNA stability or process non-coding RNAs, though its precise biological roles remain under investigation. Recent studies (as of 2024) have identified minimal twister sister-like ribozymes in the and validated twister activity in mammalian species such as the , further confirming their presence across eukaryotes. The twin ribozyme, identified in bacterial genomes, adopts a pseudoknotted fold with two active sites and catalyzes self-cleavage in intergenic regions, likely contributing to RNA turnover or regulatory circuits, with functions still being elucidated.

Evolutionary Importance

In the Origin of Life

The RNA world hypothesis posits that in the early stages of life's origin on Earth, RNA served as both the primary genetic material and the principal catalyst for biochemical reactions, predating the emergence of DNA and proteins. This concept suggests that self-replicating RNA molecules could have driven the initial Darwinian evolution by storing information and performing enzymatic functions, including replication. Evidence for this versatility comes from laboratory-evolved ribozymes capable of catalyzing RNA polymerization, demonstrating RNA's potential to replicate itself without protein assistance. Central to this hypothesis are self-replicating RNA systems that would enable the propagation of genetic variants through . Experiments by Jack Szostak's group have shown that ribozymes can catalyze the template-directed of , producing longer strands from activated monomers under conditions mimicking prebiotic environments, thus supporting the feasibility of RNA-based replication. These findings indicate that such systems could have initiated evolutionary processes by allowing RNA molecules to copy and mutate, leading to increasing complexity. Recent advances as of 2024-2025 have further bolstered the hypothesis. For instance, researchers at the Salk Institute developed an ribozyme capable of evolving improved replication fidelity through , addressing key barriers to sustained replication. Additionally, studies have demonstrated RNA-catalyzed synthesis and under prebiotic conditions, enhancing the plausibility of an RNA-based origin. The prebiotic plausibility of ribozymes hinges on their stability in primordial conditions, such as fluctuating temperatures and mineral-rich waters, where RNA's phosphodiester backbone could withstand to some extent when protected by adsorption to surfaces like clays. Ribozymes may also have played a role in metabolic origins by catalyzing the synthesis of cofactors, such as , which are remnants of early RNA-dependent biochemistry and could have facilitated energy transfer in primitive networks. The discovery of natural ribozymes, like self-splicing introns, further bolsters this by exemplifying RNA's catalytic prowess in modern biology. Despite these advances, the RNA world faces significant challenges, including RNA's chemical fragility in prebiotic settings, where and UV would degrade strands rapidly without protective mechanisms. The abiotic synthesis of RNA precursors remains a barrier, as forming under plausible conditions is inefficient and requires specific catalysts not yet fully replicated in labs. Counterarguments favoring protein primacy suggest that peptides may have preceded RNA in enabling complex catalysis, highlighting ongoing debates about the sequence of .

In Sexual Reproduction

In , group I introns play an indirect role in mating-type switching through their evolutionary contribution to the HO endonuclease gene, which is homologous to homing endonucleases encoded by these self-splicing ribozymes. The HO endonuclease initiates unidirectional switching by creating a double-strand break at the MAT locus, triggering gene conversion that copies genetic from silent cassettes (HML or HMR) to replace the expressed mating-type , thereby enabling haploid cells to alternate between a and α types and complete the sexual cycle. The mechanisms involve ribozyme-mediated self-splicing of group I to express encoded endonucleases, such as VDE (also known as PI-SceI) in related fungal systems like Ascobolus immersus, where the ribozyme activity allows production of the protein that cleaves DNA at specific sites, promoting repair via and conversion during . This process facilitates precise genetic exchange at mating-type loci, ensuring compatibility and progression through . Group I intron self-splicing, a core ribozyme function, briefly supports this by excising the intron without protein aid, using as a cofactor to join exons and release the endonuclease . Broader implications include ribozyme mobility via activity, which spreads introns unidirectionally during , altering frequencies and contributing to by favoring specific mating-type configurations across populations. This parasitic yet beneficial dynamic maintains genetic heterogeneity essential for fungal and sexual cycles.

Engineered Variants

Design and Selection Techniques

selection, often implemented through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, enables the isolation of functional ribozymes from large libraries of random sequences without prior structural knowledge. This process begins with the synthesis of a diverse pool of single-stranded molecules, typically 40–100 long, generated via or enzymatic transcription from synthetic DNA templates. The library is then subjected to iterative rounds of selection, where RNAs are exposed to a target substrate or condition that favors catalytic activity, such as ligation or cleavage; active sequences are partitioned from inactive ones, reverse-transcribed to cDNA, amplified by PCR, and transcribed back to for the next round, with stringency increased progressively to enrich for high-affinity variants. For aptazymes—ribozymes whose activity is modulated by ligand binding—SELEX variants incorporate ligand-dependent selection steps, yielding constructs like peptide-responsive ligases derived from scaffolds. Directed evolution builds on in vitro selection by introducing targeted to explore and enhance ribozyme performance, such as increasing catalytic rates or specificity. Starting from a lead ribozyme, random or site-directed mutations are introduced via error-prone PCR or , creating variant libraries that undergo or selection for improved function; for instance, iterative mutagenesis and selection have produced RNA ligase ribozymes with turnover rates exceeding 100 per hour under physiological conditions. This approach has been pivotal in allosteric ribozymes, where effector molecules trigger conformational changes to activate , as demonstrated by variants of the hammerhead ribozyme selected for small-molecule responsiveness. Computational design complements experimental methods by predicting RNA structures and functions to guide the creation of novel ribozymes, reducing reliance on trial-and-error screening. Algorithms like those in the Rosetta software suite model RNA folding and tertiary interactions using fragment assembly and energy minimization, allowing de novo design of catalytic motifs or optimization of existing scaffolds for desired activities, such as nucleotide synthesis. For example, Rosetta-based protocols have enabled the fixed-backbone redesign of RNA sequences to stabilize active conformations, achieving predicted structures with root-mean-square deviations below 3 Å from experimental models in benchmark tests. Recent advances from 2020 to 2025 have integrated these techniques for more sophisticated ribozyme engineering, including autocatalytic systems that mimic primordial synthetases. One breakthrough involves an autocatalytic ribozyme that assembles a chimeric aminoacyl-RNA synthetase through fragment aminoacylation, loop-closing ligation, and self-reinforcing cycles, enabling sustained attachment to substrates . Additionally, high-throughput platforms have facilitated the validation of novel ribozymes, identifying efficient self-cleaving motifs. These developments often start from natural scaffolds like the hammerhead ribozyme to accelerate optimization.

Applications and Recent Developments

Ribozymes have been engineered for applications, particularly through hammerhead ribozymes designed to target and cleave specific mRNA transcripts, thereby silencing pathogenic genes. In efforts against , intracellularly expressed single-chain hammerhead ribozymes have demonstrated robust suppression of by cleaving HIV-1 , with active variants showing up to 90% inhibition in models. Similarly, trans-cleaving hammerhead ribozymes have been optimized for extracellular delivery to target cancer-related genes, such as those overexpressed in tumors, enabling precise mRNA degradation without intracellular entry requirements. For instance, enhanced hammerhead variants targeting rhodopsin mRNA have exhibited improved kinetic turnover rates, supporting their potential in treating genetic disorders like , which shares mechanisms with certain cancers. These trans-cleaving designs leverage the modularity of hammerhead ribozymes for conditional in therapeutic contexts. Allosteric ribozymes, inspired by natural metabolite-responsive elements like the glmS ribozyme, have been adapted as biosensors for detecting small molecules in diagnostics. The glmS ribozyme, which undergoes self-cleavage upon binding glucosamine-6-phosphate (GlcN6P), serves as a model for engineering sensors that amplify signals through isothermal assays, achieving colorimetric detection limits as low as 1 μM for GlcN6P. These allosteric constructs integrate with amplification strategies to enhance sensitivity, making them suitable for point-of-care diagnostics where rapid metabolite detection is critical. In , ribozymes facilitate RNA processing circuits that regulate within cells, such as split ribozyme systems that detect native s and trigger orthogonal gene control. For example, ribozyme-enabled detection of RNA (RENDR) uses cellular transcripts to assemble split hammerhead ribozymes, linking RNA sensing to downstream synthetic outputs with high specificity in mammalian cells. Autocatalytic ribozyme systems have also been incorporated into models to mimic primitive replication, where encapsulation in vesicles supports studies on early life processes. Recent developments from 2020 to 2025 have expanded ribozyme applications through targeted engineering. Engineered hammerhead ribozymes directed against essential gene mRNAs in Escherichia coli have achieved significant growth inhibition, reducing bacterial proliferation by up to 70% and enhancing antibiotic efficacy like tetracycline when combined, marking the first successful targeting of vital prokaryotic transcripts. RNA-peptide coacervates have been shown to modulate ribozyme activity, with interactions inhibiting hammerhead cleavage rates by 2–3 orders of magnitude inside droplets compared to buffers, while peptide sequence variations tune catalysis for controlled synthetic environments. Metal-ion tuned structures, particularly Mg²⁺-optimized hammerhead ribozymes, select mechanically stable conformations that improve folding efficiency and catalytic persistence under physiological conditions. Additionally, high-throughput validation of novel hairpin ribozymes has expanded toolkits for RNA engineering.

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