Hairpin ribozyme

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Secondary structure of a minimal hairpin ribozyme with substrate RNA bound. Circles represent individual nucleotides and lines indicate canonical (Watson-Crick) base pairs Hairpin-ribozyme-sec-str-minimal-jpeg.jpg
Secondary structure of a minimal hairpin ribozyme with substrate RNA bound. Circles represent individual nucleotides and lines indicate canonical (Watson-Crick) base pairs

The hairpin ribozyme is a small section of RNA that can act as a ribozyme. Like the hammerhead ribozyme it is found in RNA satellites of plant viruses. It was first identified in the minus strand of the tobacco ringspot virus (TRSV) satellite RNA where it catalyzes self-cleavage and joining (ligation) reactions to process the products of rolling circle virus replication into linear and circular satellite RNA molecules. The hairpin ribozyme is similar to the hammerhead ribozyme in that it does not require a metal ion for the reaction.

Contents

Biological function

The hairpin ribozyme is an RNA motif that catalyzes RNA processing reactions essential for replication of the satellite RNA molecules in which it is embedded. These reactions are self-processing, i.e. a molecule rearranging its own structure. Both cleavage and end joining reactions are mediated by the ribozyme motif, leading to a mixture of interconvertible linear and circular satellite RNA molecules. These reactions are important for processing the large multimeric RNA molecules that are generated by rolling circle replication. At the end of the replication cycle, these large intermediates of satellite RNA replication are processed down to unit length molecules (circular or linear) before they can be packaged by viruses and carried to other cells for further rounds of replication. [1]

Folding of the hairpin ribozyme in its native tertiary structure. The ribozyme sequence is shown in grey, whilst the substrate sequence is light red. The cleavage and ligation site (dark red) is between nucleotides A-1 and G+1. Important sequences within loops A and B are shown, with black dots indicating non-Watson-Crick interactions between nucleotides. The two catalytic nucleotides are shown in green, and the critical nucleotide C25, which forms a Watson-Crick base pair with G+1 at the reaction site, is shown in blue. Hairpin-ribozyme-tertiary-structure-v2.jpg
Folding of the hairpin ribozyme in its native tertiary structure. The ribozyme sequence is shown in grey, whilst the substrate sequence is light red. The cleavage and ligation site (dark red) is between nucleotides A-1 and G+1. Important sequences within loops A and B are shown, with black dots indicating non-Watson-Crick interactions between nucleotides. The two catalytic nucleotides are shown in green, and the critical nucleotide C25, which forms a Watson-Crick base pair with G+1 at the reaction site, is shown in blue.

Natural versions of the hairpin ribozyme

In the 1980s, the hairpin ribozyme was identified in 3 naturally occurring and well-characterized sequences:

Later work in 2021 revealed almost 1000 hairpin ribozyme sequences in largely unknown organisms found in metatranscriptome data. [7] These newer sequences were hypothesized [7] to occur in organisms that, like those containing the three previously found hairpin ribozymes, use single-stranded, circular RNA genomes. The circularity of the genomes was supported experimentally, but the further nature of the organisms is not yet well studied.

Artificial versions of the hairpin ribozyme

Smaller artificial versions of the hairpin ribozyme have been developed to enable a more detailed experimental analysis of the molecule. [8] This is a commonly used strategy for separating those parts of a self-processing RNA molecule that are essential for the RNA processing reactions from those parts which serve unrelated functions. Through this process, a 50 nucleotide minimal catalytic domain and a 14 nucleotide substrate were identified. [9] Using these artificially derived sequences, a trans-acting ribozyme was developed that can catalyze the cleavage of multiple substrate molecules. This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes.

Reaction chemistry

In common with several other ribozymes and protein ribonucleases, the cleavage reaction of the hairpin ribozyme generates RNA fragments with termini consisting of a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The ligation reaction appears to be a simple reversal of cleavage, i.e. covalent joining of RNA fragments ending with a 2',3'-cyclic phosphate and a 5'-hydroxyl group to generate the ordinary 3'-5' phosphodiester linkage used in both RNA and DNA.

Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes. [10] Moreover, cleavage activity is still observed when Mg2+ is replaced by [Co(NH3)6]3+. [11] Co3+ binds NH3 so tightly in solution that NH3 does not dissociate to any appreciable extent, and therefore does not become protonated. This suggests there is no metal-catalyzed proton transfer or direct coordination to the RNA, but instead metals are only required for folding. Furthermore, in crystal structures of a ribozyme-inhibitor complex and a transition state mimic, it was shown that the three-dimensional architecture splays apart A-1 and G+1, positioning the 2'-OH of A-1 for an in-line nucleophilic attack on the scissile phosphate linkage. Additionally, G8, A38, and A9 have been suggested to play roles in the catalysis by deprotonating the 2'-OH of A-1, stabilizing the developing negative charge of the pentacoordinate phosphate oxygens, and protonating the 5'-O leaving group of G+1. [12] [13]

Structure

A representation of the 3D structure of the hairpin ribozyme. Hairpin-ribozyme-crystal-structure-UR0109.jpg
A representation of the 3D structure of the hairpin ribozyme.

The minimal hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains, each consisting of two short base paired helices separated by an internal loop. Domain A (helix 1 – loop A – helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 – loop B – helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These domains must interact with one another for catalysis to occur. [15]

When the minimal ribozyme-substrate complex is allowed to fold under conditions of low ionic strength, the two domains stack one atop the other, forming an inactive, extended structure that resembles a hairpin. [16] In order for catalysis to occur, the two domains lie parallel to one another in a fold that resembles a paperclip. In various publications, this RNA has been termed either the "paperclip" or "hairpin" ribozyme. Despite the fact that the former name has proven to be more accurate, the latter has become the commonly accepted nomenclature. In the laboratory, a functional interaction between the two domains is promoted by the addition of cations, whose positive charge suffices to overcome the electrostatic repulsion of the negatively charged RNA backbone. In nature, association of the two domains is assisted through a combination of metal ions (including Mg2+) and the presence of two additional helical domains that are not present in the minimal ribozyme-substrate complex but serve to promote proper three-dimensional folding. These additional domains stack upon helices 2 and 3, thereby promoting the association of the two functional domains through what is termed a four-way helical junction. [17]

The structure and activity of the hairpin ribozyme has been explored using a wide range of complementary experimental methods, including nucleotide replacement, functional group substitution, combinatorial selection, fluorescence spectroscopy, covalent crosslinking, NMR analysis and x-ray crystallography. These studies have been facilitated by the ability of the functional complex to self-assemble from segments made by solid phase chemical RNA synthesis, permitting the incorporation of a wide variety of modified nucleotides that are not naturally found in RNA. Together, the results of these experiments present a highly congruent picture of the catalytic cycle, i.e. how the hairpin ribozyme binds its substrate, folds into a specific three-dimensional structure, catalyzes the reaction, and releases the product(s) of the reaction. [18]

Targeted RNA cleavage and antiviral activity

Hairpin ribozymes have been modified in such a way that they can be used to target cleavage of other RNA molecules. This is possible because much of the substrate specificity of the hairpin ribozyme results from simple Watson-Crick base pairing within helices 1 and 2. [19]

One area of particular interest has been the development of hairpin ribozymes for potential therapeutic use, for example by preventing the replication of pathogenic viruses. Antiviral hairpin ribozymes have been generated and expressed within mammalian cells, and cells expressing different engineered ribozymes have been shown to be resistant to infection by HIV-1, [20] [21] hepatitis B, [22] and Sindbis virus. [23]

Related Research Articles

<span class="mw-page-title-main">RNA</span> Family of large biological molecules

Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself or by forming a template for the production of proteins. RNA and deoxyribonucleic acid (DNA) are nucleic acids. The nucleic acids constitute one of the four major macromolecules essential for all known forms of life. RNA is assembled as a chain of nucleotides. Cellular organisms use messenger RNA (mRNA) to convey genetic information that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome.

<span class="mw-page-title-main">RNA world</span> Hypothetical stage in the early evolutionary history of life on Earth

The RNA world is a hypothetical stage in the evolutionary history of life on Earth, in which self-replicating RNA molecules proliferated before the evolution of DNA and proteins. The term also refers to the hypothesis that posits the existence of this stage.

<span class="mw-page-title-main">Ribozyme</span> Type of RNA molecules

Ribozymes 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 and a biological catalyst, and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems.

Virusoids are circular single-stranded RNA(s) dependent on viruses for replication and encapsidation. The genome of virusoids consist of several hundred (200–400) nucleotides and does not code for any proteins.

Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are DNA oligonucleotides that are capable of performing a specific chemical reaction, often but not always catalytic. This is similar to the action of other biological enzymes, such as proteins or ribozymes . However, in contrast to the abundance of protein enzymes in biological systems and the discovery of biological ribozymes in the 1980s, there is only little evidence for naturally occurring deoxyribozymes. Deoxyribozymes should not be confused with DNA aptamers which are oligonucleotides that selectively bind a target ligand, but do not catalyze a subsequent chemical reaction.

<span class="mw-page-title-main">Stem-loop</span> Intramolecular base-pairing pattern in RNA and DNA

Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop. The resulting structure is a key building block of many RNA secondary structures. As an important secondary structure of RNA, it can direct RNA folding, protect structural stability for messenger RNA (mRNA), provide recognition sites for RNA binding proteins, and serve as a substrate for enzymatic reactions.

<span class="mw-page-title-main">Hammerhead ribozyme</span>

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.

<span class="mw-page-title-main">Leadzyme</span>

Leadzyme is a small ribozyme (catalytic RNA), which catalyzes the cleavage of a specific phosphodiester bond. It was discovered using an in-vitro evolution study where the researchers were selecting for RNAs that specifically cleaved themselves in the presence of lead. However, since then, it has been discovered in several natural systems. Leadzyme was found to be efficient and dynamic in the presence of micromolar concentrations of lead ions. Unlike in other small self-cleaving ribozymes, other divalent metal ions cannot replace Pb2+ in the leadzyme. Due to obligatory requirement for a lead, the ribozyme is called a metalloribozyme.

<span class="mw-page-title-main">VS ribozyme</span>

The Varkud satellite (VS) ribozyme is an RNA enzyme that carries out the cleavage of a phosphodiester bond.

<span class="mw-page-title-main">Hepatitis delta virus ribozyme</span>

The hepatitis delta virus (HDV) ribozyme is a non-coding RNA found in the hepatitis delta virus that is necessary for viral replication and is the only known human virus that utilizes ribozyme activity to infect its host. The ribozyme acts to process the RNA transcripts to unit lengths in a self-cleavage reaction during replication of the hepatitis delta virus, which is thought to propagate by a double rolling circle mechanism. The ribozyme is active in vivo in the absence of any protein factors and was the fastest known naturally occurring self-cleaving RNA at the time of its discovery.

The Lariat capping ribozyme is a ~180 nt ribozyme with an apparent resemblance to a group I ribozyme. It is found within a complex type of group I introns also termed twin-ribozyme introns. Rather than splicing, it catalyses a branching reaction in which the 2'OH of an internal residue is involved in a nucleophilic attack at a nearby phosphodiester bond. As a result, the RNA is cleaved at an internal processing site (IPS), leaving a 3'OH and a downstream product with a 3 nt lariat at its 5' end. The lariat has the first and the third nucleotide joined by a 2',5' phosphodiester bond and is referred to as 'the lariat cap' because it caps an intron-encoded mRNA. The resulting lariat cap seems to contribute by increasing the half-life of the HE mRNA, thus conferring an evolutionary advantage to the HE.

<span class="mw-page-title-main">Nucleic acid tertiary structure</span> Three-dimensional shape of a nucleic acid polymer

Nucleic acid tertiary structure is the three-dimensional shape of a nucleic acid polymer. RNA and DNA molecules are capable of diverse functions ranging from molecular recognition to catalysis. Such functions require a precise three-dimensional structure. While such structures are diverse and seemingly complex, they are composed of recurring, easily recognizable tertiary structural motifs that serve as molecular building blocks. Some of the most common motifs for RNA and DNA tertiary structure are described below, but this information is based on a limited number of solved structures. Many more tertiary structural motifs will be revealed as new RNA and DNA molecules are structurally characterized.

<span class="mw-page-title-main">Nucleic acid secondary structure</span>

Nucleic acid secondary structure is the basepairing interactions within a single nucleic acid polymer or between two polymers. It can be represented as a list of bases which are paired in a nucleic acid molecule. The secondary structures of biological DNAs and RNAs tend to be different: biological DNA mostly exists as fully base paired double helices, while biological RNA is single stranded and often forms complex and intricate base-pairing interactions due to its increased ability to form hydrogen bonds stemming from the extra hydroxyl group in the ribose sugar.

<span class="mw-page-title-main">Hypercycle (chemistry)</span> Cyclic sequence of self-reproducing single cycles

In chemistry, a hypercycle is an abstract model of organization of self-replicating molecules connected in a cyclic, autocatalytic manner. It was introduced in an ordinary differential equation (ODE) form by the Nobel Prize in Chemistry winner Manfred Eigen in 1971 and subsequently further extended in collaboration with Peter Schuster. It was proposed as a solution to the error threshold problem encountered during modelling of replicative molecules that hypothetically existed on the primordial Earth. As such, it explained how life on Earth could have begun using only relatively short genetic sequences, which in theory were too short to store all essential information. The hypercycle is a special case of the replicator equation. The most important properties of hypercycles are autocatalytic growth competition between cycles, once-for-ever selective behaviour, utilization of small selective advantage, rapid evolvability, increased information capacity, and selection against parasitic branches.

<span class="mw-page-title-main">Retroviral ribonuclease H</span>

The retroviral ribonuclease H is a catalytic domain of the retroviral reverse transcriptase (RT) enzyme. The RT enzyme is used to generate complementary DNA (cDNA) from the retroviral RNA genome. This process is called reverse transcription. To complete this complex process, the retroviral RT enzymes need to adopt a multifunctional nature. They therefore possess 3 of the following biochemical activities: RNA-dependent DNA polymerase, ribonuclease H, and DNA-dependent DNA polymerase activities. Like all RNase H enzymes, the retroviral RNase H domain cleaves DNA/RNA duplexes and will not degrade DNA or unhybridized RNA.

<span class="mw-page-title-main">Twister ribozyme</span> Ribozyme capable of self-cleavage

The twister ribozyme is a catalytic RNA structure capable of self-cleavage. The nucleolytic activity of this ribozyme has been demonstrated both in vivo and in vitro and has one of the fastest catalytic rates of naturally occurring ribozymes with similar function. The twister ribozyme is considered to be a member of the small self-cleaving ribozyme family which includes the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), and glmS ribozymes.

<span class="mw-page-title-main">RNA hydrolysis</span>

RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a hydroxyl group at the 2’ position. This feature makes RNA chemically unstable compared to DNA, which does not have this 2’ -OH group and thus is not susceptible to base-catalyzed hydrolysis.

<span class="mw-page-title-main">Twister sister ribozyme</span> RNA structure

The twister sister ribozyme (TS) is an RNA structure that catalyzes its own cleavage at a specific site. In other words, it is a self-cleaving ribozyme. The twister sister ribozyme was discovered by a bioinformatics strategy as an RNA Associated with Genes Associated with Twister and Hammerhead ribozymes, or RAGATH.

<span class="mw-page-title-main">Hatchet ribozyme</span> Self-cleaving ribozyme

Background: The hatchet ribozyme is an RNA structure that catalyzes its own cleavage at a specific site. In other words, it is a self-cleaving ribozyme. Hatchet ribozymes were discovered by a bioinformatics strategy as RNAs Associated with Genes Associated with Twister and Hammerhead ribozymes, or RAGATH.

Retrozymes are a family of retrotransposons first discovered in the genomes of plants but now also known in genomes of animals. Retrozymes contain a hammerhead ribozyme (HHR) in their sequences, although they do not possess any coding regions. Retrozymes are nonautonomous retroelements, and so borrow proteins from other elements to move into new regions of a genome. Retrozymes are actively transcribed into covalently closed circular RNAs and are detected in both polarities, which may indicate the use of rolling circle replication in their lifecycle.

References

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