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.     

Partition of aminoacyl-tRNA synthetases in two different structural classes dating back to early metabolism: Implications for the origin of the genetic code and the nature of protein sequences

We describe, on the molecular level, a possible fuzzy and primordial translation apparatus capable of synthesizing polypeptides from nucleic acids in a world containing a mixture of coevolving molecules of RNA and proteins already arranged in metabolic cycles (including cofactors). Close attention is paid to template-free systems because they are believed to be the immediate ancestors of this primordial translation apparatus. The two classes of amnoacyl-tRNA synthetases (aaRSs), as seen today, are considered as the remnants of such a simple imprecise translation apparatus and are used as guidelines for the construction of the model. Earlier theoretical work by Bedian on a related system is invoked to show how specificity and stability could have been achieved automatically and rather quickly, starting from such an imprecise system, i.e., how the encoded synthesis of proteins could have appeared. Because of the binary nature of the underlying proto-code, the first genetically encoded proteins would then have been alternating copolymers with a high degree of degeneracy, but not random. Indeed, a clear signal for alternating hydrophobic and hydrophilic residues in present-day protein sequences can be detected. Later evolution of the genetic code would have proceeded along lines already discussed by Crick. However, in the initial stages, the translation apparatus proposed here is in fact very similar to the one postulated by Woese, only here it is given a molecular framework. This hypothesis departs from the paradigm of the RNA world in that it supposes that the origin of the genetic code occurred after the apparition of some functional (statistical) proteins first. Implications for protein design are also discussed.     

The origin of the genetic code and protein synthesis

A model for a parallel evolution of the genetic code and protein synthesis is presented. The main tenet of this model is that the genetic code, that is, a correspondence between nucleotide and aminoacid coding units, originated from sequence-specific interaction between abiotically synthesized polynucleotides and polypeptides. A sequence-specific binding between oligonucleotides and oligopeptides is supported by experimental findings. Moreover, it is parsimonious enough to be consistent with the relatively simple chemistry of a primordial environment. Proximity between peptides and RNA increased the rate of formation of ester bonds between them. This lead to the accumulation of sequence-specific polypeptide-polynucleotide pairs, that is, of primordial-loaded tRNA. Condensation of short polypeptides into longer products could be catalyzed by a sequence-specific juxtaposition of loaded tRNA over complementary RNA, originating the core of protein synthesis. The accumulation of useful encoded products, for example, catalysts for tRNA loading (primordial aminoacyl-tRNA synthetases) or stabilizers of tRNA-mRNA interactions (primordial ribosomes), permitted the subsequent evolution of protein synthesis and of the genetic code to their mature form. This occurred via a parallel reduction in length of the interacting polynucleotides and polypeptides. Thus, it maintained the correct reading frame of mRNA from the preceding stages of evolution. Received: 27 September 1996 / Accepted: 17 May 1997     

The driving force for molecular evolution of translation

It is widely argued that protein synthesis evolved out of an RNA world, in which catalytic and other biological functions now carried out by proteins were performed by RNAs. However, it is not clear what selective advantage would have provided the driving force for evolution of a primitive translation apparatus, because of the unlikelihood that rudimentary polypeptides would have contributed sufficiently useful biological functions. Here, I suggest that the availability of even simple peptides could have significantly enlarged the otherwise limited structure space of RNA. In other words, translation initially evolved not to create a protein world, but to extend the structural, and therefore the functional, capabilities of the RNA world. Observed examples of substantial structural rearrangements in RNA that are induced by binding of peptides and other small molecules support this possibility.     

Codon Size Reduction as the Origin of the Triplet Genetic Code

The genetic code appears to be optimized in its robustness to missense errors and frameshift errors. In addition, the genetic code is near-optimal in terms of its ability to carry information in addition to the sequences of encoded proteins. As evolution has no foresight, optimality of the modern genetic code suggests that it evolved from less optimal code variants. The length of codons in the genetic code is also optimal, as three is the minimal nucleotide combination that can encode the twenty standard amino acids. The apparent impossibility of transitions between codon sizes in a discontinuous manner during evolution has resulted in an unbending view that the genetic code was always triplet. Yet, recent experimental evidence on quadruplet decoding, as well as the discovery of organisms with ambiguous and dual decoding, suggest that the possibility of the evolution of triplet decoding from living systems with non-triplet decoding merits reconsideration and further exploration. To explore this possibility we designed a mathematical model of the evolution of primitive digital coding systems which can decode nucleotide sequences into protein sequences. These coding systems can evolve their nucleotide sequences via genetic events of Darwinian evolution, such as point-mutations. The replication rates of such coding systems depend on the accuracy of the generated protein sequences. Computer simulations based on our model show that decoding systems with codons of length greater than three spontaneously evolve into predominantly triplet decoding systems. Our findings suggest a plausible scenario for the evolution of the triplet genetic code in a continuous manner. This scenario suggests an explanation of how protein synthesis could be accomplished by means of long RNA-RNA interactions prior to the emergence of the complex decoding machinery, such as the ribosome, that is required for stabilization and discrimination of otherwise weak triplet codon-anticodon interactions.     

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     

Photosynthesis and the Origin of Life

The origin and evolution of photosynthesis is considered to be the key to the origin of life. This eliminates the need for a soup as the synthesis of the bioorganics are to come from the fixation of carbon dioxide and nitrogen. No soup then no RNA world or Protein world. Cyanobacteria have been formed by the horizontal transfer of green sulfur bacterial photoreaction center genes by means of a plasmid into a purple photosynthetic bacterium. The fixation of carbon dioxide is considered to have evolved from a reductive dicarboxylic acid cycle (Chloroflexus) which was then followed by a reductive tricarboxylic acid cycle (Chlorobium) and finally by the reductive pentose phosphate cycle (Calvin cycle). The origin of life is considered to have occurred in a hot spring on the outgassing early earth. The first organisms were self-replicating iron-rich clays which fixed carbon dioxide into oxalic and other dicarboxylic acids. This system of replicating clays and their metabolic phenotype then evolved into the sulfide rich region of the hotspring acquiring the ability to fix nitrogen. Finally phosphate was incorporated into the evolving system which allowed the synthesis of nucleotides and phospholipids. If biosynthesis recapitulates biopoesis, then the synthesis of amino acids preceded the synthesis of the purine and pyrimidine bases. Furthermore the polymerization of the amino acid thioesters into polypeptides preceded the directed polymerization of amino acid esters by polynucleotides. Thus the origin and evolution of the genetic code is a late development and records the takeover of the clay by RNA.     

A speculation on the origin of protein synthesis

It is suggested that protein synthesis may have begun without even a primitive ribosome if the primitive tRNA could take up two configurations and could bind to the messenger RNA with five base-pairs instead of the present three. This idea would impose base sequence restriction on the early messages and on the early genetic code such that the first four amino acids coded were glycine, serine, aspartic acid and aspargine. A possible mechanism is suggested for the polymerization of the early message.This paper is dedicated to the memory of Dr. Aharon Katzir.     

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.     

Oxytricha as a modern analog of ancient genome evolution

Several independent lines of evidence suggest that the modern genetic system was preceded by the 'RNA world' in which RNA genes encoded RNA catalysts. Current gaps in our conceptual framework of early genetic systems make it difficult to imagine how a stable RNA genome may have functioned and how the transition to a DNA genome could have taken place. Here we use the single-celled ciliate, Oxytricha, as an analog to some of the genetic and genomic traits that may have been present in organisms before and during the establishment of a DNA genome. Oxytricha and its close relatives have a unique genome architecture involving two differentiated nuclei, one of which encodes the genome on small, linear nanochromosomes. While its unique genomic characteristics are relatively modern, some physiological processes related to the genomes and nuclei of Oxytricha may exemplify primitive states of the developing genetic system.     

Hypotheses on the establishment of a genetic code and transfer of information from proteinoids to nucleic acids

An hypothesis is proposed in which the specificity of interaction between an aminoacid and a nucleotide sequence of a tRNA would be enhanced by a ternary association with a specific proteinoid. These strict relations would have led to the present genetic code that we know. It is also proposed that the origin of the enzymatic activity of the primitive proteinoids would have arisen from the presence of different substrates during polymerisation, which would have favored specific sequences of aminoacids by forming more stable complexes with them, corresponding to the lowest free enthalpy. The information included in the aminoacid sequences of the proteinoids would have been transferred to messenger type RNA, according to a mechanism reverse of that for the present process for protein synthesis, and then to DNA.     

Structural Phylogenomics Retrodicts the Origin of the Genetic Code and Uncovers the Evolutionary Impact of Protein Flexibility

The genetic code shapes the genetic repository. Its origin has puzzled molecular scientists for over half a century and remains a long-standing mystery. Here we show that the origin of the genetic code is tightly coupled to the history of aminoacyl-tRNA synthetase enzymes and their interactions with tRNA. A timeline of evolutionary appearance of protein domain families derived from a structural census in hundreds of genomes reveals the early emergence of the ‘operational’ RNA code and the late implementation of the standard genetic code. The emergence of codon specificities and amino acid charging involved tight coevolution of aminoacyl-tRNA synthetases and tRNA structures as well as episodes of structural recruitment. Remarkably, amino acid and dipeptide compositions of single-domain proteins appearing before the standard code suggest archaic synthetases with structures homologous to catalytic domains of tyrosyl-tRNA and seryl-tRNA synthetases were capable of peptide bond formation and aminoacylation. Results reveal that genetics arose through coevolutionary interactions between polypeptides and nucleic acid cofactors as an exacting mechanism that favored flexibility and folding of the emergent proteins. These enhancements of phenotypic robustness were likely internalized into the emerging genetic system with the early rise of modern protein structure.     

Selection, history and chemistry: the three faces of the genetic code.

The genetic code might be a historical accident that was fixed in the last common ancestor of modern organisms. 'Adaptive', 'historical' and 'chemical' arguments, however, challenge such a 'frozen accident' model. These arguments propose that the current code is somehow optimal, reflects the expansion of a more primitive code to include more amino acids, or is a consequence of direct chemical interactions between RNA and amino acids, respectively. Such models are not mutually exclusive, however. They can be reconciled by an evolutionary model whereby stereochemical interactions shaped the initial code, which subsequently expanded through biosynthetic modification of encoded amino acids and, finally, was optimized through codon reassignment. Alternatively, all three forces might have acted in concert to assign the 20 'natural' amino acids to their present positions in the genetic code.     

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 Early Phases of Genetic Code Origin: Conjectures on the Evolution of Coded Catalysis

A review of the most significant contributions on the early phases of genetic code origin is presented. After stressing the importance of the key intermediary role played in protein synthesis, by peptidyl-tRNA, which is attributed with a primary function in ancestral catalysis, the general lines leading to the codification of the first amino acids in the genetic code are discussed. This is achieved by means of a model of protoribosome evolution which sees protoribosome as the central organiser of ancestral biosynthesis and the mediator of the encounter between compounds (metabolite-pre-tRNAs) and catalysts (peptidyl-pre-tRNAs). The encounter between peptidyl-pre-tRNA catalysts in protoribosome is favoured by metabolic pre-mRNAs and later resulted (given the high temperature at which this evolution is supposed to have taken place) in the evolution of mRNAs with codons of the type GNS. These mRNAs codified only for those amino acids that the coevolution theory of genetic code origin sees as the precursors of all other amino acids. Some aspects of the model here discussed might be rendered real by the transfer-messenger RNA molecule (tmRNA) which is here considered a molecular fossil of ancestral protein synthesis.     

Proteins, exons and molecular evolution

The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.     

Speculations on the evolution of the genetic code IV the evolution of the aminoacyl-tRNA synthetases

An evolutionary scheme is postulated in which a primitive code, involving only guanine and cytosine, would code for glycine(GG.), alanine(GC), arginine(CG.) and proline(CC). There evolves from this primitive code families of related amino acids as the code expands. The evolution of the aminiacyl-tRNA synthetases are considered to be indicators for the evolution of the genetic code. The postulated model for the evolution of the genetic code is used to give an evolutionary interpretation to the recent work on the structure and sequences of the aminoacyl-tRNA synthetases.     

Early assignments of the genetic code dependent upon protein structure

Orgel (1972) has suggested that polynucleotides with sequences of alternating purine/pyrimidine are likely to have predominated in prebiotic conditions. Therefore, in any early template-directed protein synthesis, the number of available codons would have been limited. However, for any self-organizing system to survive and propagate, some feedback must occur from the products of the synthesis to the control of the synthetic procedure itself; i.e. the protein synthesized should have catalysed some step in the initiation of template-directed synthesis. A given protein structure with a characteristic conformation and function would be optimal for the product of such a synthesis, and this in turn would limit the number of nucleotide sequences of those available able to give rise to a functioning synthetic assembly. A possible candidate for such an early polypeptide is the ferredoxin group of proteins and it is shown that with the present-day code the corresponding nucleotides do have a high percentage of alternating purine/pyrimidine sequences. Hence these combined restraints on the primitive synthetic machinery would direct the possible assignments of the genetic code helping to explain its regularity and universality.     

A possible origin of RNA catalysis in multienzyme complexes

Numerous attempts have recently been made to ascribe a preeminent role to RNA enzymes in primitive life systems. A model is proposed in which coenzyme-dependent RNA enzymes were initially organized in multienzyme complexes featuring (1) the continuous attachment of substrates to CoA-like carriers, as in fatty acid synthesis; and (2) the ordering of RNA enzymes via mRNA-like instructional strands. In this format, RNA enzymes would not have been required to recognized and specifically bind soluble substrates. The enzymes in this case may have required far less complexity than contemporary protein enzymes and thus less genetic information for their synthesis. An analogy is made between the proposed scheme and the protein translation mechanism, for which it may have been an evolutionary precursor.