Ribozyme
In a recent open access paper in the online journal, Life, a Tokyo University of Science team led by Professor Koji Tamura of the Department of Biological Science and Technology, has given support to the 'RNA World' hypothesis that explains the beginnings of self-replicating organisms as self-catalysing RNA assemblies known as 'ribozymes'.
Ribozymes have the ability to catalyse the production of new ribozymes, so overcoming the 'problem' for abiogenesis that proteins are needed to catalyse the assembly of DNA but DNA is needed to create proteins, leading to a classic 'chicken and egg' paradox. Since ribozymes don't need proteins, this solves the problem.
How Professor Tamura' team did this is the subject of a Tokyo University of Science news release:
RNA's Hidden Potential: New Study Unveils Its Role in Early Life and Future Bioengineering
Study sheds light on the molecular evolution of RNA and its potential applications in nanobiotechnology.
The beginning of life on Earth and its evolution over billions of years continue to intrigue researchers worldwide. The central dogma or the directional flow of genetic information from a deoxyribose nucleic acid (DNA) template to a ribose nucleic acid (RNA) transcript, and finally into a functional protein, is fundamental to cellular structure and functions. DNA functions as the blueprint of the cell and carries genetic information required for the synthesis of functional proteins. Conversely, proteins are required for the synthesis of DNA. Therefore, whether DNA emerged first or protein, continues to remain a matter of debate.
This molecular version of the "chicken and egg" question led to the proposition of an "RNA World." RNAs in the form of 'ribozymes' or RNA enzymes carry genetic information similar to DNA and also possess catalytic functions like proteins. The discovery of ribozymes further fueled the RNA World hypothesis where RNA served dual functions of "genetic information storage" and "catalysis," facilitating primitive life activities solely by RNA. While modern ribosomes are a complex of RNAs and proteins, ribozymes during early evolutionary stages may have been pieced together through the assembly of individual functional RNA units.
To test this hypothesis, Professor Koji Tamura, along with his team of researchers at the Department of Biological Science and Technology, Tokyo University of Science, conducted a series of experiments to decode the assembly of functional ribozymes. For this, they designed an artificial ribozyme, R3C ligase, to investigate how individual RNA units come together to form a functional structure. Giving further insight into their work published on 17 April 2024, in Life, Prof. Tamura states:Within ribosomes, which are the site of protein synthesis, RNA units assemble to function as Peptidyl Transferase Center (PTC) in a way such that they form a scaffold for the recruitment of amino acids (individual components of a peptide/protein) attached to tRNAs (featured in Nature magazine (https://www.nature.com/articles/d41586-023-00574-4)). This is an important insight into the evolutionary history of protein synthesis systems, but it is not sufficient to trace the evolutionary pathway based on the RNA World hypothesis.The R3C ligase is a ribozyme that catalyzes the formation of a 3',5'-phosphodiester linkage between two RNA molecules. We modified the structure by adding specific domains that can interact with various effectors.
Professor Koji Tamura, co-corresponding author
Department of Biological Science and Technology
Tokyo University of Science, Tokyo, Japan.
To explore if the elongation of RNA, achieved by linking individual RNA units together, is regulated allosterically, the researchers altered the structure of the R3C ligase. They did this by incorporating short RNA sequences that bind adenosine triphosphate (ATP), a vital energy carrier molecule in cells, into the ribozyme. The team noted that R3C ligase activity was dependent on the concentration of ATP, with higher activity observed at higher concentrations of ATP. Further, an increase in the melting temperature (Tm value) indicated that the binding of ATP to R3C ligase stabilized the structure, which likely influenced its ligase activity.
Similarly, on fusing an L-histidine-binding RNA sequence to the ribozyme, they noted an increase in ligase activity at increasing concentrations of histidine (a key amino acid). Notably, the increase in activity was specific to increasing concentrations of ATP or histidine; no changes were observed in response to other nucleotide triphosphates or amino acids. These findings suggest that ATP and histidine act as effector molecules that trigger structural conformational changes in the ribozyme, which further influence enzyme stability and activity.
ATP is the central energy carrier of the cell which supports numerous molecular processes, while, histidine is the most common amino acid found in the active site of enzymes, and maintains their acid-base chemistry. Given, the important roles of ATP and histidine in RNA interactions and molecular functions, these results provide novel insights into the role of RNA in early evolution, including the origin of the genetic code. Furthermore, engineered ribozymes such as the one developed in this study hold significant promise in a myriad of applications including targeted drug delivery, therapeutics, nano-biosensors, enzyme engineering, and synthesis of novel enzymes with uses in various industrial processes.
Overall, this study can offer insights into how the transition from the RNA World to the modern "DNA/Protein World" occurred. A fundamental understanding of the RNA World in turn, can enhance their use in real-life applications.
This study will lead to the elucidation of the process of 'allostericity-based acquisition of function and cooperativity' in RNA evolution. The RNA-RNA interactions, RNA-amino acid interactions, and allostericity applied in this research can guide the fabrication of arbitrary RNA nanostructures, with various applications.
Professor Koji Tamura.
AbstractIt seems then that the power of RNA to self-catalyse and to kick-start an evolutionary process that led to progressive improvements in the earliest cells hasn't before been realised, so, given the molecules that would have been available on Earth and the conditions that would have been found in the right locations, the beginnings of 'life' was nothing more than the operation of basic laws of chemistry and physics, with an evolutionary process being an inevitable result, leading eventually to DNA as RNA's data store.
During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.
1. Introduction
All life on Earth is based on the concept of “central dogma”, the unidirectional flow of genetic information [1]. When considering the evolution of genetic information transmission systems on primitive Earth, it is difficult to imagine that the complex systems of DNA, RNA, and proteins of modern life suddenly appeared. DNA is necessary for protein synthesis, and proteins are necessary to synthesize DNA and RNA. Therefore, which was generated first, nucleic acids or proteins? There is some debate as to whether “central dogma” is in fact correct in the origin of life. Biological reactions have long been thought to be controlled by enzymes and catalysts comprising proteins. An enzyme (reverse transcriptase) that synthesizes DNA using the base sequences of RNA as a template has been discovered [2], proving that RNA carries genetic information similar to that of DNA. Furthermore, the catalytic activity of RNA has been demonstrated without the involvement of proteins [3,4]. Thus, RNA has both “genetic information storage” and “catalytic” functions. The discovery of ribozymes led to the proposal of the RNA world hypothesis, which states that in the early developmental stages of life on Earth, RNA possessed both the genetic information storage function of DNA and the catalytic function of proteins; thus, the activities of living organisms were carried out solely by RNA [5].
Although modern ribosomes are a complex of RNAs and proteins, they would have assembled during early evolution by the association and joining of small functional RNA units [6]. Noller and coworkers’ experiment suggested that the peptidyl transferase center (PTC) on the ribosome is composed of RNA [7], and the crystal structure of the PTC clearly proved it [8,9]. PTC is formed by two symmetrically arranged tRNA-like units [10,11,12,13,14], and certain combinations of the symmetrical segments are capable of mediating peptide bond formation [15,16], even between two aminoacyl-minihelices (primordial tRNAs), tethered by the dimeric scaffold [16]. In the evolution of rRNA, introns may have provided the means to ligate many of these pieces together. A survey of rRNA intron sequences, locations, and structural characteristics across the three major life domains suggests that rRNA gene loci may have served as evolutionary nurseries for intron formation and diversification [17]. In addition, based on recent scientific advances in Coronaviridae, the transition between RNA- to DNA-based life RNA is suggested to be driven by an evolutionary relationship between RNA polymerases, RNA exonuclease, and RNA methyltransferases [18].
It would be extremely important to conduct experiments that demonstrate an aspect of the RNA world: the concatenation of RNA domains to give rise to new functional RNAs. Ribozymes have various catalytic abilities such as cleavage, ligation, and splicing. In this study, we used the R3C ligase ribozyme (73 nucleotides [nt]) [19], which catalyzes a nucleophilic attack by a 3′-hydroxyl of the substrate on a 5′-α-phosphorus of the triphosphates of the ribozyme itself to form a 3′-5′-phosphodiester bond (Figure 1A). Eigen’s concept of a “hypercycle” argues that a length of less than 100 nucleotides is required for self-replication without necessitating an error-correcting mechanism [20]. In addition, the length of RNA synthesized in the clay mineral environment that was expected to have existed on primitive Earth is less than 50 nt [21]. Consequently, Kurihara et al. reduced the size of the R3C ligase ribozyme from 73 to 46 nt [22]. However, the ligase activity of RNA minimized to 46 nt is only 0.8% of the activity of the full-length R3C ligase ribozyme (73 nt). To restore the activity of the reduced ligase, Tanizawa et al. performed an experiment focusing on the loop portion of a minimized ribozyme [23]. RNA stabilizes its structure by forming kissing loops [24,25,26]. Using this interaction, two RNAs with complementary sequences in the loop regions of R3C ligase variants were prepared, and the mixed RNA system showed significantly increased ligation activity compared to that of the individually performed reactions. Multiple functions can be achieved via kissing loop interactions, suggesting their versatility in the evolution of RNA [27].
Allosteric regulation of protein enzymes involves allosteric binding sites, located apart from the active site of the enzyme [28]. Binding of the effector molecule to the allosteric binding site induces conformational changes in the protein structure that alter the catalytic rate of the enzyme. In the case of hemoglobin [29], the binding of oxygen to one of the subunits induces conformational changes that are relayed to the other subunits, making them more able to bind oxygen by raising their affinity for this molecule [30]. Similarly, during the evolution of the RNA world, it is probable that during the RNA elongation process, a short RNA would have fused with the domain that interacted with a specific compound (effector), resulting in an RNA whose activity was regulated by and dependent on a specific effector (Figure 1B). In the L1 ligase ribozyme [31], a ribozyme is created by fusing an ATP-binding aptamer RNA to create an allosteric ligase ribozyme that increases ligase activity in an ATP concentration-dependent manner [32]. Using the R3C ligase, which is smaller than the L1 ligase, we aimed to develop general allosteric ribozymes that are independent of the effector molecule type.
Figure 1. (A) Compositions of the R3C ligase ribozyme (73-mer, top) and that of the reduced form, R3C-A (46-mer, bottom). They are shown in black. The 5′-6-FAM-labeled ligation substrate (12-mer) is shown in green. The stem loop regions of the original R3C ligase ribozyme are designated as “Hammer” and “Grip”. (B) Schematic presentation of the evolution process of ribozymes. During the RNA elongation process, a short RNA would have fused with other RNA domains that interacted with specific compounds (effectors), resulting in an RNA whose activity regulation was dependent on the specific effector. Each circle represents a nucleotide comprising RNA.
Akatsu, Y.; Mutsuro-Aoki, H.; Tamura, K.
Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.
Life 2024, 14, 520. https://doi.org/10.3390/life14040520
Copyright: © [year] The authors.
Published by [publisher]. Open access.
Reprinted under a Creative Commons Attribution 4.0 International license (CC BY 4.0)
In many ways, living organisms are still RNA-based since DNA needs to be transcribed into messenger RNA (mRNA) as the functional medium for the information to build proteins in the RNA-based ribosomes (the descendants of the early ribozymes) and transfer RNA (tRNA) binding to amino acids to splice them into the protein chain in the correct order as determined by the mRNA transcribed from the DNA.
And abiogenesis is simply a matter of chemistry and physics, with no need for God-magic or mysterious 'life' substance at any stage.
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