Relics from the RNA World

An RNA world is widely accepted as a probable stage in the early evolution of life. Two implications are that proteins have gradually replaced RNA as the main biological catalysts and that RNA has not taken on any major de novo catalytic function after the evolution of protein synthesis, that is, there is an essentially irreversible series of steps RNA → RNP → protein. This transition, as expected from a consideration of catalytic perfection, is essentially complete for reactions when the substrates are small molecules. Based on these principles we derive criteria for identifying RNAs in modern organisms that are relics from the RNA world and then examine the function and phylogenetic distribution of RNA for such remnants of the RNA world. This allows an estimate of the minimum complexity of the last ribo-organism—the stage just preceding the advent of genetically encoded protein synthesis. Despite the constraints placed on its size by a low fidelity of replication (the Eigen limit), we conclude that the genome of this organism reached a considerable level of complexity that included several RNA-processing steps. It would include a large protoribosome with many smaller RNAs involved in its assembly, pre-tRNAs and tRNA processing, an ability for recombination of RNA, some RNA editing, an ability to copy to the end of each RNA strand, and some transport functions. It is harder to recognize specific metabolic reactions that must have existed but synthetic and bio-energetic functions would be necessary. Overall, this requires that such an organism maintained a multiple copy, double-stranded linear RNA genome capable of recombination and splicing. The genome was most likely fragmented, allowing each ``chromosome' to be replicated with minimum error, that is, within the Eigen limit. The model as developed serves as an outgroup to root the tree of life and is an alternative to using sequence data for inferring properties of the earliest cells. Received: 14 January 1997 / Accepted: 19 May 1997     

Polynucleotide replication coupled to protein synthesis: A possible mechanism for the origin of life

A mechanism is suggested for the replication under primitive conditions of long polynucleotides by the sequential incorporation of sequences related to those of modern transfer RNAs. It is proposed that replication of such molecules became established as the result of a replicative advantage arising from the concomitant linkage together of amino acids to form polypeptides. Initially these polypeptides may have been of random sequence. Selection of primitive tRNAs in which the amino acid and anticodon stem sequences were rotaionally symmetrical could have led to specific, anticodon-directed aminoacylation and fixation of the genetic code along the lines suggested by Hopfield. (Hopfield, 1978). The primitive replication-coupled system would then have been able to synthesize specific proteins containing one amino acid residue for each primitive tRNA incorporated during replication. The end result of this line of evolution is postulated to have been a nucleoprotein structure resembling the ribosome. The primitive system would then have been able to give rise directly to triplet-coded protein synthesis. Some recent RNA sequence data are discussed which are consistent with derivation of modern protein synthesis from the primitive replication-coupled mechanism.     

The Path from the RNA World

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA → RNP → protein, the first proteins in the ``breakthrough organism' (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the ``introns-first' theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a ``eukaryotic-like' genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome → eukaryotic-like DNA genome → prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome → eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA → RNP → protein. A likely molecular mechanism, ``plasmid transfer,' is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history. Received: 14 January 1997 / Accepted: 19 May 1997     

Molecular Mimicry Between Protein and tRNA

Mimicry is a sophisticated development in animals, fish, and plants that allows them to fool others by imitating a shape or color for diverse purposes, such as to prey, evade, lure, pollinate, or threaten. This is not restricted to the macro-world, but extends to the micro-world as molecular mimicry. Recent advances in structural and molecular biology uncovered a set of translation factors that resembles a tRNA shape and, in one case, even mimics a tRNA function for deciphering the genetic code. Nature must have evolved this art of molecular mimicry between protein and ribonucleic acid by using different protein structures until the translation factors sat in the cockpit of a ribosome machine, on behalf of tRNA, and achieved diverse actions. Structural, functional, and evolutionary aspects of molecular mimicry will be discussed. Received: 9 January 2001 / Accepted: 22 March 2001     

A Sequential Scenario for the Origin of Biological Chirality

A sequential model is proposed regarding the origin of biological chirality. Three major stages are presumed: a symmetry breaking (prebiotic chiral disruption in enantiomeric mixtures of monomers), a chiral amplification (prebiotic increase of the chiral character of the monomers affected first by the symmetry breaking), and a chiral expansion (proto biological increase of the chiral character and spread of the chirality to molecules which were less affected by prebiotic chiralizations). As a symmetry-breaking mechanism, the model proposed by Deutsch (1991) is used, which involves a dissymmetric exposure of amino acids (AA) to ultraviolet circularly polarized light (UV-CPL) on evaporative seashores. It is presumed that the chiral amplification, up to a protobiologic significance, was influenced by a periodic overlapping of two abiotic events, a synchronization between tidal-based hydrous–anhydrous cycles, and littoral asymmetric photolysis cycles. This long-term astronomic asymmetry acted around 3.8–4.2 billion years ago and was unique to the Earth in our solar system. It is also presumed that the abiotic symmetry breaking is heterogenous, that only a few l-AAs were used in the beginning, and that the chirality expanded later to all 20 AAs based on a coevolutionary strategy of the genetic code and on a physiological relationship between AAs. In this scenario the d-chirality of pentoses in polynucleotides was attributed to both d-pentose/l-AA relationships and to a structural evolution. Received: 10 May 1996 / Accepted: 13 August 1996     

The Genetic Code: What Is It Good For? An Analysis of the Effects of Selection Pressures on Genetic Codes

How did the ``universal' genetic code arise? Several hypotheses have been put forward, and the code has been analyzed extensively by authors looking for clues to selection pressures that might have acted during its evolution. But this approach has been ineffective. Although an impressive number of properties has been attributed to the universal code, it has been impossible to determine whether selection on any of these properties was important in the code's evolution or whether the observed properties arose as a consequence of selection on some other characteristic. Therefore we turned the question around and asked, what would a genetic code look like if it had evolved in response to various different selection pressures? To address this question, we constructed a genetic algorithm. We found first that selecting on a particular measure yields codes that are similar to each other. Second, we found that the universal code is far from minimized with respect to the effects of mutations (or translation errors) on the amino acid compositions of proteins. Finally, we found that the codes that most closely resembled real codes were those generated by selecting on aspects of the code's structure, not those generated by selecting to minimize the effects of amino acid substitutions on proteins. This suggests that the universal genetic code has been selected for a particular structure—a structure that confers an important flexibility on the evolution of genes and proteins—and that the particular assignments of amino acids to codons are secondary. Received: 29 December 1998 / Accepted: 8 July 1999     

Relationships Among Isoacceptor tRNAs Seems to Support the Coevolution Theory of the Origin of the Genetic Code

A new method for looking at relationships between nucleotide sequences has been used to analyze divergence both within and between the families of isoaccepting tRNA sets. A dendrogram of the relationships between 21 tRNA sets with different amino acid specificities is presented as the result of the analysis. Methionine initiator tRNAs are included as a separate set. The dendrogram has been interpreted with respect to the final stage of the evolutionary pathway with the development of highly specific tRNAs from ambiguous molecular adaptors. The location of the sets on the dendrogram was therefore analyzed in relation to hypotheses on the origin of the genetic code: the coevolution theory, the physicochemical hypothesis, and the hypothesis of ambiguity reduction of the genetic code. Pairs of 16 sets of isoacceptor tRNAs, whose amino acids are in biosynthetic relationships, occupied contiguous positions on the dendrogram, thus supporting the coevolution theory of the genetic code. Received: 4 May 1998 / Accepted: 11 July 1998     

On the Evolution of Redundancy in Genetic Codes

We simulate a deterministic population genetic model for the coevolution of genetic codes and protein-coding genes. We use very simple assumptions about translation, mutation, and protein fitness to calculate mutation-selection equilibria of codon frequencies and fitness in a large asexual population with a given genetic code. We then compute the fitnesses of altered genetic codes that compete to invade the population by translating its genes with higher fitness. Codes and genes coevolve in a succession of stages, alternating between genetic equilibration and code invasion, from an initial wholly ambiguous coding state to a diversified frozen coding state. Our simulations almost always resulted in partially redundant frozen genetic codes. Also, the range of simulated physicochemical properties among encoded amino acids in frozen codes was always less than maximal. These results did not require the assumption of historical constraints on the number and type of amino acids available to codes nor on the complexity of proteins, stereochemical constraints on the translational apparatus, nor mechanistic constraints on genetic code change. Both the extent and timing of amino-acid diversification in genetic codes were strongly affected by the message mutation rate and strength of missense selection. Our results suggest that various omnipresent phenomena that distribute codons over sites with different selective requirements—such as the persistence of nonsynonymous mutations at equilibrium, the positive selection of the same codon in different types of sites, and translational ambiguity—predispose the evolution of redundancy and of reduced amino acid diversity in genetic codes. Received: 21 December 2000 / Accepted: 12 March 2001     

Aminoacylation of RNA Minihelices: Implications for tRNA Synthetase Structural Design and Evolution

Abstract

The genetic code is based on the aminoacylation of tRNA with amino acids catalyzed by the aminoacyl-tRNA synthetases. The synthetases are constructed from discrete domains and all synthetases possess a core catalytic domain that catalyzes amino acid activation, binds the acceptor stem of tRNA, and transfers the amino acid to tRNA. Fused to the core domain are additional domains that mediate RNA interactions distal to the acceptor stem. Several synthetases catalyze the aminoacylation of RNA oligonucleotide substrates that recreate only the tRNA acceptor stems. In one case, a relatively small catalytic domain catalyzes the aminoacylation of these substrates independent of the rest of the protein. Thus, the active site domain may represent a primordial synthetase in which polypeptide insertions that mediate RNA acceptor stem interactions are tightly integrated with determinants for aminoacyl adenylate synthesis. The relationship between nucleotide sequences in small RNA oligonucleotides and the specific amino acids that are attached to these oligonucleotides could constitute a second genetic code.     

On the RNA World: Evidence in Favor of an Early Ribonucleopeptide World

A highly complex RNA world, as is sometimes presented in view of the widespread and diversified use of RNA enzymes, would have encountered many difficulties in passing to a world with catalysis mediated by proteins. These difficulties can be overcome by postulating a very early relationship between the nucleotide and the amino acid components. In particular, after asserting that some characteristics expressed by (nucleotide) coenzymes in catalysis are easier to understand if a close and early relationship between these coenzymes and amino acids is hypothesized, a model is presented for the origin of the enzyme–coenzyme complex. This model is essentially based on an intermediate formed by a tRNA-like molecule covalently linked to a polypeptide. The model attributes the majority of the catalytic role in the ribonucleoprotein world to the latter complex and thus it takes into account the birth of the key intermediate in the origin of protein synthesis—namely, peptidyl-tRNA, which would have otherwise been extremely difficult to select. The predictions of the model are discussed along with its robustness, using the data derived from the study of intermediary metabolism and those from molecular biology. Finally, the appearance of the genetic code in the late phase of the ribonucleopeptide world is discussed. Received: 13 January 1997 / Accepted: 25 July 1997     

Evolved RNA Secondary Structure and the Rooting of the Universal Tree of Life

The origin and diversification of RNA secondary structure were traced using cladistic methods. Structural components were coded as polarized and ordered multi-state characters, following a model of character state transformation outlined by considerations in statistical mechanics. Several classes of functional RNA were analyzed, including ribosomal RNA (rRNA). Considerable phylogenetic signal was present in their secondary structure. The intrinsically rooted phylogenies reconstructed from evolved RNA structure depicted those derived from nucleic acid sequence at all taxonomical levels, and grouped organisms in concordance with traditional classification, especially in the archaeal and eukaryal domains. Natural selection appears therefore to operate early in the information flow that originates in sequence and ends in an adapted phenotype. When examining the hierarchical classification of the living world, phylogenetic analysis of secondary structure of the small and large rRNA subunits reconstructed a universal tree of life that branched in three monophyletic groups corresponding to Eucarya, Archaea, and Bacteria, and was rooted in the eukaryotic branch. Ribosomal characters involved in the translational cycle could be easily traced and showed that transfer RNA (tRNA) binding domains in the large rRNA subunit evolved concurrently with the rest of the rRNA molecule. Results suggest it is equally parsimonious to consider that ancestral unicellular eukaryotes or prokaryotes gave rise to all extant life forms and provide a rare insight into the early evolution of nucleic acid and protein biosynthesis. Received: 13 September 2000 / Accepted: 27 August 2001     

Cell growth inhibition by sequence-specific RNA minihelices.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.     

Metabolic complexity in the RNA world and implications for the origin of protein synthesis

Summary A model is presented for the evolution of metabolism and protein synthesis in a primitive, acellular RNA world. It has been argued previously that the ability to perform metabolic functions logically must have preceded the evolution of a message-dependent protein synthetic machinery and that considerable metabolic complexity was achieved by ribo-organisms (i.e., organisms in which both genome and enzymes are comprised of RNA). The model proposed here offers a mechanism to account for the gradual development of sophisticated metabolic activities by ribo-organisms and explains how such metabolic complexity would lead subsequently to the synthesis of genetically encoded polypeptides. RNA structures ancestral to modern ribosomes, here termed metabolosomes, are proposed to have functioned as organizing centers that coordinated, using base-pairing interactions, the order and nature of adaptor-mounted substrate/catalyst interactions in primitive metabolic pathways. In this way an ancient genetic code for metabolism is envisaged to have predated the specialized modern genetic code for protein synthesis. Thus, encoded amino acids initially would have been used, in conjunction with other encoded metabolites, as building blocks for biosynthetic pathways, a role that they retain in the metabolism of contemporary organisms. At a later stage the encoded amino acids would have been condensed together on similar RNA metabolosome structures to form the first genetically determined, and therefore biologically meaningful, polypeptides. On the basis of codon distributions in the modern genetic code it is argued that the first proteins to have been synthesized and used by ribo-organisms were predominantly hydrophobic and likely to have performed membrane-related functions (such as forming simple pore structures), activities essential for the evolution of membrane-enclosed cells.     

Origins of tmRNA: the missing link in the birth of protein synthesis?

The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.     

The origin of the protein synthesis mechanism

The origin and development of the protein synthesis mechanism is considered in four successive steps. The genetic code is supposed to be controlled by the relative amount (availability) of various amino acids and nucleotides on the one hand, and utility on each amino acid in the polypeptide. on the other hand. Thus, more simple (inutile) and abundant amino acids tended to correspond to codons which were rich in the less frequent base species, G and C. Features of primitive tRNA in the discrimination of amino acid are discussed. Primitive tRNA is proposed to have a discriminator site for amino acid and, separated from it, an anticodon site for interaction with nucleotides. A hypothetical course of subdivision of various nucleic acid species is proposed. In the scheme, mRNA and ribosomal RNA (rRNA) were derived from more primitive insoluble RNA. DNA appeared in the late, not first, step of the development. Several other aspects of evolutionary development of the whole protein synthesis mechanism, e.g., role of the discriminator site on primitive tRNA, modification and subdivision of code catalogue into a more precise specification of amino acids, and possible primordial interactions between tRNA and tRNA-binding sites on insoluble rRNA, are discussed.     

Phylogenetic Affinity of Mitochondria of Euglena gracilis and Kinetoplastids Using Cytochrome Oxidase I and hsp60

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5′ and 3′ untranslated transcribed sequences and lack a 3′ poly[A] tail. The COI gene does not require uridine insertion/deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [α-³²P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3′ end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences. Received: 5 March 1996 / Accepted: 31 July 1996     

Hypothesis: Emergence of Translation as a Result of RNA Helicase Evolution

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998     

The Level and Landscape of Optimization in the Origin of the Genetic Code

We consider a model of the origin of genetic code organization incorporating the biosynthetic relationships between amino acids and their physicochemical properties. We study the behavior of the genetic code in the set of codes subject both to biosynthetic constraints and to the constraint that the biosynthetic classes of amino acids must occupy only their own codon domain, as observed in the genetic code. Therefore, this set contains the smallest number of elements ever analyzed in similar studies. Under these conditions and if, as predicted by physicochemical postulates, the amino acid properties played a fundamental role in genetic code organization, it can be expected that the code must display an extremely high level of optimization. This prediction is not supported by our analysis, which indicates, for instance, a minimization percentage of only 80%. These observations can therefore be more easily explained by the coevolution theory of genetic code origin, which postulates a role that is important but not fundamental for the amino acid properties in the structuring of the code. We have also investigated the shape of the optimization landscape that might have arisen during genetic code origin. Here, too, the results seem to favor the coevolution theory because, for instance, the fact that only a few amino acid exchanges would have been sufficient to transform the genetic code (which is not a local minimum) into a much better optimized code, and that such exchanges did not actually take place, seems to suggest that, for instance, the reduction of translation errors was not the main adaptive theme structuring the genetic code.