Archaeal-bacterial chimeric RNase P RNAs: towards understanding RNA's architecture, function and evolution

The higher protein content of archaeal RNase P (1 RNA+4 proteins) compared to the bacterial homologue (1 RNA+1 protein) correlates with a large loss of RNA-alone activity (i.e., in the absence of protein cofactors). Here we show, for the first time, that a catalytic (C) domain of an archaeal RNase P RNA (P RNA) can functionally replace the Escherichia coli C domain in a chimeric P RNA, to provide the essential RNase P function in E. coli cells. This adaptation was achieved by 1) three minor alterations in the archaeal C domain, 2) restoration of the L9-P1 interdomain contact that is found in bacterial and archaeal type A RNAs, and 3) installation of another interdomain contact (L18-P8) that is present in bacterial but absent in archaeal P RNAs. We conclude 1) that the C domains of bacterial and archaeal P RNAs of type A have been largely conserved since the evolutionary separation of bacteria and archaea, and 2) that the L18-P8 RNA-RNA contact has been replaced with protein-protein contacts in archaeal RNase P. Function of the chimeric P RNA in E. coli required overexpression of the E. coli RNase P protein to increase the RNA's reduced cellular levels; this was attributed to enhanced degradation of the chimeric P RNA.     

RNA Relics and Origin of Life

A number of small RNA sequences, located in different non-coding sequences and highly preserved across the tree of life, have been suggested to be molecular fossils, of ancient (and possibly primordial) origin. On the other hand, recent years have revealed the existence of ubiquitous roles for small RNA sequences in modern organisms, in functions ranging from cell regulation to antiviral activity. We propose that a single thread can be followed from the beginning of life in RNA structures selected only for stability reasons through the RNA relics and up to the current coevolution of RNA sequences; such an understanding would shed light both on the history and on the present development of the RNA machinery and interactions. After presenting the evidence (by comparing their sequences) that points toward a common thread, we discuss a scenario of genome coevolution (with emphasis on viral infectious processes) and finally propose a plan for the reevaluation of the stereochemical theory of the genetic code; we claim that it may still be relevant, and not only for understanding the origin of life, but also for a comprehensive picture of regulation in present-day cells.     

Evolutionary History of Bioessential Elements Can Guide the Search for Life in the Universe

Our understanding of life in the universe comes from one sample, life on Earth. Current and next-generation space missions will target exoplanets as well as planets and moons in our own solar system with the primary goal of detecting, interpreting and characterizing indications of possible biological activity. Thus, understanding life's fundamental characteristics is increasingly critical for detecting and interpreting potential biological signatures elsewhere in the universe. Astrobiologists have outlined the essential roles of carbon and water for life, but we have yet to decipher the rules governing the evolution of how living organisms use bioessential elements. Does the suite of life's essential chemical elements on Earth constitute only one possible evolutionary outcome? Are some elements so essential for biological functions that evolution will select for them despite low availability? How would this play out on other worlds that have different relative element abundances? When we look for life in the universe, or the conditions that could give rise to life, we must learn how to recognize it in extremely different chemical and environmental conditions from those on Earth. We argue that by exposing self-organizing biotic chemistries to different combinations of abiotic materials, and by mapping the evolutionary history of metalloenzyme biochemistry onto geological availabilities of metals, alternative element choices that are very different from life's present-day molecular structure might result. A greater understanding of the paleomolecular evolutionary history of life on Earth will create a predictive capacity for detecting and assessing life's existence on worlds where alternate evolutionary paths might have been taken.     

Genome Evolution from Random Ligation of RNAs of Autocatalytic Sets

The evolutionary origin of the genome remains elusive. Here, I hypothesize that its first iteration, the protogenome, was a multi-ribozyme RNA. It evolved, likely within liposomes (the protocells) forming in dry-wet cycling environments, through the random fusion of ribozymes by a ligase and was amplified by a polymerase. The protogenome thereby linked, in one molecule, the information required to seed the protometabolism (a combination of RNA-based autocatalytic sets) in newly forming protocells. If this combination of autocatalytic sets was evolutionarily advantageous, the protogenome would have amplified in a population of multiplying protocells. It likely was a quasispecies with redundant information, e.g., multiple copies of one ribozyme. As such, new functionalities could evolve, including a genetic code. Once one or more components of the protometabolism were templated by the protogenome (e.g., when a ribozyme was replaced by a protein enzyme), and/or addiction modules evolved, the protometabolism became dependent on the protogenome. Along with increasing fidelity of the RNA polymerase, the protogenome could grow, e.g., by incorporating additional ribozyme domains. Finally, the protogenome could have evolved into a DNA genome with increased stability and storage capacity. I will provide suggestions for experiments to test some aspects of this hypothesis, such as evaluating the ability of ribozyme RNA polymerases to generate random ligation products and testing the catalytic activity of linked ribozyme domains.     

Chemistry Constraints on the Origin of Life

This analysis starts from the view that prebiotic chemical evolution, leading to the first forms of life on earth, was based on a series of sequential steps, each determined by its own contingent initial conditions. This view is opposed to the more established one, which sees the origin of life as a series of preordered series of events, where each one is deterministically caused by the previous one and causally determines the next one. Some of the main constraints of chemistry that affect such prebiotic chemical evolution are examined. The notion of contingency is seen as a very important organizing process subjected to chemistry, whereby contingency also responds to a certain degree of determinism. Kinetic control, as another determinant and constraint of the prebiotic evolutionary process, can be critically important and, at a certain point of the chemical evolutionary process, kinetic control in the form of catalysis will become essential. Simple peptides can be considered as the first catalysts, at least for the condensation of peptide bonds. The concentration threshold for prebiotic reactions is often not taken into account in the literature, particularly in the field of the prebiotic RNA world. In addition, this shortcoming can make the entire prebiotic RNA world construction shaky and unreliable, including the “myth” of the perennial self-replication of an RNA macromolecule. The general question of self-replication and the problem of homochirality are also briefly discussed. Although these chemical constraints may hinder the reconstruction of life as it is now in the laboratory, their understanding can be useful and even essential for devising a synthetic alternative route to functional macromolecules and to their metabolic interactions.     

Activation of the glmS Ribozyme Confers Bacterial Growth Inhibition

The ever‐growing number of pathogenic bacteria resistant to treatment with antibiotics call for the development of novel compounds with as‐yet unexplored modes of action. Here, we demonstrate the in vivo antibacterial activity of carba‐α‐d ‐glucosamine (CGlcN). In this mode of action study, we provide evidence that CGlcN‐mediated growth inhibition is due to glmS ribozyme activation, and we demonstrate that CGlcN hijacks an endogenous activation pathway, hence utilizing a prodrug mechanism. This is the first report describing antibacterial activity mediated by activating the self‐cleaving properties of a ribozyme. Our results open the path towards a compound class with an entirely novel and distinct molecular mechanism.     

Tecto‐GIRz: Engineered Group I Ribozyme the Catalytic Ability of Which Can Be Controlled by Self‐Dimerization

RNA is a promising biomaterial for self‐assembly of nano‐sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA‐based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto‐GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.     

RNA Aminoacylation Mediated by Sequential Action of Two Ribozymes and a Nonactivated Amino Acid

In the transition from the RNA world to the modern DNA/protein world, RNA‐catalyzed aminoacylation might have been a key step towards early translation. A number of ribozymes capable of aminoacylating their own 3′ termini have been developed by in vitro selection. However, all of those catalysts require a previously activated amino acid—typically an aminoacyl‐AMP—as substrate. Here we present two ribozymes connected by intermolecular base pairing and carrying out the two steps of aminoacylation: ribozyme 1 loads nonactivated phenylalanine onto its phosphorylated 5′ terminus, thereby forming a high‐energy mixed anhydride. Thereafter, a complex of ribozymes 1 and 2 is formed by intermolecular base pairing, and the “activated” phenylalanine is transferred from the 5′ terminus of ribozyme 1 to the 3′ terminus of ribozyme 2. This kind of simple RNA aminoacylase complex was engineered from previously selected ribozymes possessing the two required activities. RNA aminoacylation with a nonactivated amino acid as described here is advantageous to RNA world scenarios because initial amino acid activation by an additional reagent (in most cases, ATP) and an additional ribozyme would not be necessary.     

Development and Applications of Coarse-Grained Models for RNA

In contrast to proteins, much less attention has been focused on the development of computational models for describing RNA molecules, which are being recognized as playing key roles in many cellular functions. Current atomically detailed force fields are not accurate enough to capture the properties of even simple nucleic acid constructs. In this article, we review our efforts to develop coarse-grained (CG) models that capture the underlying physics for the particular length scale of interest. Two models are discussed. One of them is the three interaction site (TIS) model, in which each nucleotide is represented by three beads corresponding to sugar, phosphate, and base. The other is the self-organized polymer (SOP) model, in which each nucleotide is represented as a single interaction center. Applications of the TIS model to study the complexity of hairpin formation and the effects of crowding in a shifting equilibrium between two conformations in human telomerase pseudoknot are described. The work on crowding illustrates a direct link to the activity of telomerase. We use the SOP model to describe the response of the Tetrahymena ribozyme to force. The simulated unfolding pathways agree well with single molecule pulling experiments. We also review predictions for the unfolding pathways for the Azoarcus ribozyme. The success of the CG applications to describe dynamics in RNA gives hope that more complex processes involving RNA-protein interactions can be tackled using variants of the proposed models.     

Aptamer to ribozyme: the intrinsic catalytic potential of a small RNA

The discovery of RNA-based catalysis 23 years ago dramatically changed the way biologists and biochemists thought of RNA. In the recent past, several ribozymes structures have provided some answers as to how catalysis is accomplished and how it relates to RNA structure and folding. However, there is still little information as to how catalytic activity evolved. Here we show that the small malachite green-binding aptamer has intrinsic catalytic potential that can be realized by designing the proper substrate. The charge distribution within the RNA binding pocket stabilizes the transition state of an ester hydrolysis reaction and thus accelerates the overall reaction. The results suggest that electrostatic forces can contribute significantly to RNA-based catalysis. Moreover, even simple RNA structures that have not been selected for catalytic properties can have a basic catalytic potential if they encounter the right substrate. This provides a possible starting point for the molecular evolution of more complex ribozymes.     

Mapping the Conformational Landscape of the Neutral Network of RNA Sequences That Connect Two Functional Distinctly Different Ribozymes

During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000 , 289, 448–452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network.     

Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme

The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl₂ concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg²⁺ concentration required for substrate cleavage. An inverse correlation was found between the MgCl₂ concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg²⁺‐induced structural transitions. The results suggest that the solution environment has various effects on the Mg²⁺ interactions involved in the catalysis and global folding of the ribozyme.     

Coenzyme Autocatalytic Network on the Surface of Oil Microspheres as a Model for the Origin of Life

Coenzymes are often considered as remnants of primordial metabolism, but not as hereditary molecules. I suggest that coenzyme-like molecules (CLMs) performed hereditary functions before the emergence of nucleic acids. Autocatalytic CLMs modified (encoded) surface properties of hydrocarbon microspheres, to which they were anchored, and these changes enhanced autocatalysis and propagation of CLMs. Heredity started from a single kind of self-reproducing CLM, and then evolved into more complex coenzyme autocatalytic networks containing multiple kinds of CLMs. Polymerization of CLMs on the surface of microspheres and development of template-based synthesis is a potential evolutionary path towards the emergence of nucleic acids.     

Formation of L Amino Acids and D Sugars,and Amplification of their Enantioexcesses in Aqueous Solutions,Under Simulated Prebiotic Conditions

The Murchison meteorite delivered five α-methyl amino acids to Earth with small excesses of the L enantiomer; later, additional examples were found and such compounds were also found in other meteorites. I describe our work using them under prebiotic conditions to form normal proteinogenic amino acids with an excess of the L enantiomers, and to amplify such excesses to dominant concentration in solution. I also extend this work to show how D sugars, such as D -ribose, can have been formed and amplified in solution. I also show the high concentration amplifications of D -nucleosides that can be obtained under credible prebiotic conditions. The simple theory of such amplifications, and corrections resulting from solvation effects, are described along with modern ideas of the source of such α-methyl amino acids in meteorites.