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.     

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.     

In the Beginning was a Mutualism - On the Origin of Translation

The origin of translation is critical for understanding the evolution of life, including the origins of life. The canonical genetic code is one of the most dominant aspects of life on this planet, while the origin of heredity is one of the key evolutionary transitions in living world. Why the translation apparatus evolved is one of the enduring mysteries of molecular biology. Assuming the hypothesis, that during the emergence of life evolution had to first involve autocatalytic systems which only subsequently acquired the capacity of genetic heredity, we propose and discuss possible mechanisms, basic aspects of the emergence and subsequent molecular evolution of translation and ribosomes, as well as enzymes as we know them today. It is possible, in this sense, to view the ribosome as a digital-to-analogue information converter. The proposed mechanism is based on the abilities and tendencies of short RNA and polypeptides to fold and to catalyse biochemical reactions. The proposed mechanism is in concordance with the hypothesis of a possible chemical co-evolution of RNA and proteins in the origin of the genetic code or even more generally at the early evolution of life on Earth. The possible abundance and availability of monomers at prebiotic conditions are considered in the mechanism. The hypothesis that early polypeptides were folding on the RNA scaffold is also considered and mutualism in molecular evolutionary development of RNA and peptides is favoured.     

Revisiting the Physico-Chemical Hypothesis of Code Origin: An Analysis Based on Code-Sequence Coevolution in a Finite Population

The origin of the genetic code marked a major transition from a plausible RNA world to the world of DNA and proteins and is an important milestone in our understanding of the origin of life. We examine the efficacy of the physico-chemical hypothesis of code origin by carrying out simulations of code-sequence coevolution in finite populations in stages, leading first to the emergence of ten amino acid code(s) and subsequently to 14 amino acid code(s). We explore two different scenarios of primordial code evolution. In one scenario, competition occurs between populations of equilibrated code-sequence sets while in another scenario; new codes compete with existing codes as they are gradually introduced into the population with a finite probability. In either case, we find that natural selection between competing codes distinguished by differences in the degree of physico-chemical optimization is unable to explain the structure of the standard genetic code. The code whose structure is most consistent with the standard genetic code is often not among the codes that have a high fixation probability. However, we find that the composition of the code population affects the code fixation probability. A physico-chemically optimized code gets fixed with a significantly higher probability if it competes against a set of randomly generated codes. Our results suggest that physico-chemical optimization may not be the sole driving force in ensuring the emergence of the standard genetic code.     

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10⁻¹⁰ per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC.     

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.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

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.     

Optimization Models and the Structure of the Genetic Code

The codon assignment of the quasi-universal genetic code can be assumed to have resulted from the evolutionary pressures that prevailed when the code was still evolving. Here, we review studies of the structure of the genetic code based on optimization models. We also review studies that, from the structure of the code, attempt to derive aspects of the primordial circumstances in which the genetic code froze. Different rationales are summarized, compared with experimental data, discussed in the context of the transition from a RNA world to a DNA-protein world, and linked to the emergence of the last universal common ancestor.     

Does the Genetic Code Have A Eukaryotic Origin?

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core "house-keeping" functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables-GC and purine contents-of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern-the symmetric pattern-where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes.     

RNA templating of molecular assembly and covalent modification patterning in early molecular evolution and modern biosystems

The Direct RNA Template (DRT) hypothesis proposes that an early stage of genetic code evolution involved RNA molecules acting as stereochemical recognition templates for assembly of specific amino acids in sequence-ordered arrays, providing a framework for directed covalent peptide bond formation. It is hypothesized here that modern biological precedents may exist for RNA-based structural templating with functional analogies to hypothetical DRT systems. Beyond covalent molecular assembly, an extension of the DRT concept can include RNA molecules acting as dynamic structural template guides for the specific non-covalent assembly of multi-subunit complexes, equivalent to structural assembly chaperones. However, despite numerous precedents for RNA molecules acting as scaffolds for protein complexes, true RNA-mediated assembly chaperoning appears to be absent in modern biosystems. Another level of function with parallels to a DRT system is possible if RNA structural motifs dynamically guided specific patterns of catalytic modifications within multiple target sites in a pre-formed polymer or macromolecular complex. It is suggested that this type of structural RNA templating could logically play a functional role in certain areas of biology, one of which is the glycome of complex organisms. If any such RNA templating processes are shown to exist, they would share no necessary evolutionary relationships with events during early molecular evolution, but may promote understanding of the practical limits of biological RNA functions now and in the ancient RNA World. Awareness of these formal possibilities may also assist in the current search for functions of extensive non-coding RNAs in complex organisms, or for efforts towards artificial rendering of DRT systems.     

Physico-chemical constraints connected with the coding properties of the genetic system

New insights on the origin of the genetic code, based on the analysis of the physico-chemical properties of its molecular constituents (RNA and amino acids), are reported in this paper. We point out a symmetry in the genetic code table and show that it can be explained by the nature of the anticodon-codon interaction. The importance of the strength of this interaction is examined and a correlation is found between the free-energy change (DeltaG(0)) of anticodon-codon association and the volume of the corresponding amino acids. This correlation is investigated in conjunction with the well-known one linking the hydrophobicity of the anticodons with that of the amino acids. We show that they can be considerated separately and that the energy vs. volume correlation may be explained by the process implicating the peptide bond formation between two successive amino acids during translation. This interpretation is supported by a statistical pattern of bases (purines or pyrimidines), observed in present coding genes, and by considerations involving the availability of the different kinds of amino acids. Finally, we try to explain the hydrophobicity correlation when reconstructing the events at the time of the so-called "RNA World". The whole of our investigation shows that the genetic code might be sufficiently robust to exist without the participation of pre-existing proteins, and that this robustness is a consequence of the physico-chemical properties of the four bases of the genetic system.     

Genetic Variation for Life History Sensitivity to Seasonal Warming in Arabidopsis thaliana

Climate change has altered life history events in many plant species; however, little is known about genetic variation underlying seasonal thermal response. In this study, we simulated current and three future warming climates and measured flowering time across a globally diverse set of Arabidopsis thaliana accessions. We found that increased diurnal and seasonal temperature (1°–3°) decreased flowering time in two fall cohorts. The early fall cohort was unique in that both rapid cycling and overwintering life history strategies were revealed; the proportion of rapid cycling plants increased by 3–7% for each 1° temperature increase. We performed genome-wide association studies (GWAS) to identify the underlying genetic basis of thermal sensitivity. GWAS identified five main-effect quantitative trait loci (QTL) controlling flowering time and another five QTL with thermal sensitivity. Candidate genes include known flowering loci; a cochaperone that interacts with heat-shock protein 90; and a flowering hormone, gibberellic acid, a biosynthetic enzyme. The identified genetic architecture allowed accurate prediction of flowering phenotypes (R² > 0.95) that has application for genomic selection of adaptive genotypes for future environments. This work may serve as a reference for breeding and conservation genetic studies under changing environments.     

Genetic Hotels for the Standard Genetic Code: Evolutionary Analysis Based upon Novel Three-Dimensional Algebraic Models

Herein, we rigorously develop novel 3-dimensional algebraic models called Genetic Hotels of the Standard Genetic Code (SGC). We start by considering the primeval RNA genetic code which consists of the 16 codons of type RNY (purine-any base-pyrimidine). Using simple algebraic operations, we show how the RNA code could have evolved toward the current SGC via two different intermediate evolutionary stages called Extended RNA code type I and II. By rotations or translations of the subset RNY, we arrive at the SGC via the former (type I) or via the latter (type II), respectively. Biologically, the Extended RNA code type I, consists of all codons of the type RNY plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The Extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. Since the dimensions of remarkable subsets of the Genetic Hotels are not necessarily integer numbers, we also introduce the concept of algebraic fractal dimension. A general decoding function which maps each codon to its corresponding amino acid or the stop signals is also derived. The Phenotypic Hotel of amino acids is also illustrated. The proposed evolutionary paths are discussed in terms of the existing theories of the evolution of the SGC. The adoption of 3-dimensional models of the Genetic and Phenotypic Hotels will facilitate the understanding of the biological properties of the SGC.     

The RNA/protein symmetry hypothesis: experimental support for reverse translation of primitive proteins

Although the "RNA-world" theory, or the RNA-first theory is renowned for a promising theory of biogenesis, it is also possible that both RNAs and proteins have coevolved forming a stable metabolic complex from the very beginning. I investigated this possibility assuming that the genetic information flowed symmetrically in the era of the origin of life, i.e. the primitive translation machinery worked in both directions (from RNA to protein and from protein to RNA). According to this RNA/protein symmetry theory, the genetic information would have come from existing cellular proteins via reverse translation. This process would have been completed in a short period of time without searching an enormous RNA sequence space. Furthermore, reverse translation would have ensured biological continuity; proteins that were essential for cellular metabolism would have been utilized in the same way as before the protein sequence information would have been transferred into the RNA sequences. I also propose a possible mechanism for the process of reverse translation. The reverse translation would proceed in the 3' to 5' direction using a set of at least 20 reverse transfer RNAs (rtRNAs) that can recognize their specific amino acid residue and carry their corresponding codon. A source of genetic information would be a primary sequence of a protein molecule. Several basic steps of reverse translation were demonstrated using rtRNA(Arg).