The 3 31 Nucleotide Minihelix tRNA Evolution Theorem and the Origin of Life.

There are no theorems (proven theories) in the biological sciences. We propose that the 3 31 nt minihelix tRNA evolution theorem be universally accepted as one. The 3 31 nt minihelix theorem completely describes the evolution of type I and type II tRNAs from ordered precursors (RNA repeats and inverted repeats). Despite the diversification of tRNAome sequences, statistical tests overwhelmingly support the theorem. Furthermore, the theorem relates the dominant pathway for the origin of life on Earth, specifically, how tRNAomes and the genetic code may have coevolved. Alternate models for tRNA evolution (i.e., 2 minihelix, convergent and accretion models) are falsified. In the context of the pre-life world, tRNA was a molecule that, via mutation, could modify anticodon sequences and teach itself to code. Based on the tRNA sequence, we relate the clearest history to date of the chemical evolution of life. From analysis of tRNA evolution, ribozyme-mediated RNA ligation was a primary driving force in the evolution of complexity during the pre-life-to-life transition. TRNA formed the core for the evolution of living systems on Earth.

The Evolution of Life Is a Road Paved with the DNA Quadruplet Symmetry and the Supersymmetry Genetic Code.

Symmetries have not been completely determined and explained from the discovery of the DNA structure in 1953 and the genetic code in 1961. We show, during 10 years of investigation and research, our discovery of the Supersymmetry Genetic Code table in the form of 2 × 8 codon boxes, quadruplet DNA symmetries, and the classification of trinucleotides/codons, all built with the same physiochemical double mirror symmetry and Watson-Crick pairing. We also show that single-stranded RNA had the complete code of life in the form of the Supersymmetry Genetic Code table simultaneously with instructions of codons' relationship as to how to develop the DNA molecule on the principle of Watson-Crick pairing. We show that the same symmetries between the genetic code and DNA quadruplet are highly conserved during the whole evolution even between phylogenetically distant organisms. In this way, decreasing disorder and entropy enabled the evolution of living beings up to sophisticated species with cognitive features. Our hypothesis that all twenty amino acids are necessary for the origin of life on the Earth, which entirely changes our view on evolution, confirms the evidence of organic natural amino acids from the extra-terrestrial asteroid Ryugu, which is nearly as old as our solar system.

Modern and prebiotic amino acids support distinct structural profiles in proteins.

The earliest proteins had to rely on amino acids available on early Earth before the biosynthetic pathways for more complex amino acids evolved. In extant proteins, a significant fraction of the 'late' amino acids (such as Arg, Lys, His, Cys, Trp and Tyr) belong to essential catalytic and structure-stabilizing residues. How (or if) early proteins could sustain an early biosphere has been a major puzzle. Here, we analysed two combinatorial protein libraries representing proxies of the available sequence space at two different evolutionary stages. The first is composed of the entire alphabet of 20 amino acids while the second one consists of only 10 residues (ASDGLIPTEV) representing a consensus view of plausibly available amino acids through prebiotic chemistry. We show that compact conformations resistant to proteolysis are surprisingly similarly abundant in both libraries. In addition, the early alphabet proteins are inherently more soluble and refoldable, independent of the general Hsp70 chaperone activity. By contrast, chaperones significantly increase the otherwise poor solubility of the modern alphabet proteins suggesting their coevolution with the amino acid repertoire. Our work indicates that while both early and modern amino acids are predisposed to supporting protein structure, they do so with different biophysical properties and via different mechanisms.

Trinucleotide k-circular codes II: Biology.

A code X is (⩾k)-circular if every concatenation of words from X that admits, when read on a circle, more than one partition into words from X, must contain at least k+1 words. In other words, the reading frame retrieval is guaranteed for any concatenation of up to k words from X. A code that is (⩾k)-circular for all integers k is said to be circular. Any code is (⩾0)-circular and it turns out that a code of trinucleotides is circular as soon as it is (⩾4)-circular. A code is k-circular if it is (⩾k)-circular and not (⩾k+1)-circular. The theoretical aspects of trinucleotide k-circular codes have been developed in a companion article (Michel et al., 2022). Trinucleotide circular codes always retrieve the reading frame, leaving no ambiguous sequences. On the contrary, trinucleotide k-circular codes, for k∈{0,1,2,3} all have ambiguous sequences, for which the reading frame cannot always be retrieved. However, such a trinucleotide k-circular code is still able to retrieve the reading frame for a number of sequences, thereby exhibiting a partial circularity property. We describe this combinatorial property for each class of trinucleotide k-circular codes with k∈{0,1,2,3}. The circularity, i.e. the reading frame retrieval, is an ordinary property in genes. In order to consider the different cases of ambiguous sequences, we derive a new and general formula to measure the reading frame loss, whatever the trinucleotide k-circular code. This formula allows us to study the evolution of any trinucleotide k-circular code of (maximal) cardinality 20 to the genetic code, based on the reading frame retrieval property. We apply this approach to analyse the evolution of the trinucleotide circular code X observed in genes to the genetic code. The (⩾1)-circular codes of maximal size 20 necessarily have the same number of each nucleotide, specifically 15=3⋅20/4. This balanceness property can also be achieved by trinucleotide codes of cardinality 4,8,12 and 16. We call such trinucleotide codes balanced. We develop a general mathematical method to compute the number of balanced trinucleotide codes of each size, which also applies to self-complementary trinucleotide codes. We establish and quantify a relation between this balanceness property and the self-complementarity property. The combinatorial hierarchy of trinucleotide k-circular codes is updated with the growth function results. The numbers of amino acids coded by the trinucleotide k-circular codes are given for the cases maximal, minimal, self-complementary k-, (k,k,k)- and self-complementary (k,k,k)-circular.

In Vitro Evolution Reveals Noncationic Protein-RNA Interaction Mediated by Metal Ions.

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from an approximately 1010 library of partially randomized sequences, all composed of ten prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with molecular dynamics simulations uncovers qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.

The shaping of a molecular linguist: How a career studying DNA energetics revealed the language of molecular communication.

My personal and professional journeys have been far from predictable based on my early childhood. Owing to a range of serendipitous influences, I miraculously transitioned from a rebellious, apathetic teenage street urchin who did poorly in school to a highly motivated, disciplined, and ambitious academic honors student. I was the proverbial "late bloomer." Ultimately, I earned my PhD in biophysical chemistry at Yale, followed by a postdoc fellowship at Berkeley. These two meccas of thermodynamics, coupled with my deep fascination with biology, instilled in me a passion to pursue an academic career focused on mapping the energy landscapes of biological systems. I viewed differential energetics as the language of molecular communication that would dictate and control biological structures, as well as modulate the modes of action associated with biological functions. I wanted to be a "molecular linguist." For the next 50 years, my group and I used a combination of spectroscopic and calorimetric techniques to characterize the energy profiles of the polymorphic conformational space of DNA molecules, their differential ligand-binding properties, and the energy landscapes associated with mutagenic DNA damage recognition, repair, and replication. As elaborated below, the resultant energy databases have enabled the development of quantitative molecular biology through the rational design of primers, probes, and arrays for diagnostic, therapeutic, and molecular-profiling protocols, which collectively have contributed to a myriad of biomedical assays. Such profiling is further justified by yielding unique energy-based insights that complement and expand elegant, structure-based understandings of biological processes.

Enzyme catalysis prior to aromatic residues: Reverse engineering of a dephospho-CoA kinase.

The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.

Redox Biochemistry of the Genetic Code.

New findings on the chemistry of the amino acids, their role in protein folding, and their sequential primordial introduction have uncovered concealed causalities in genetic code evolution. The genetically encoded amino acids successively provided (i) membrane anchors, (ii) halophilic protein folds, (iii) mesophilic protein folds, (iv) metal ligation, and (v) antioxidation.

The Relation Between k-Circularity and Circularity of Codes.

A code X is k-circular if any concatenation of at most k words from X, when read on a circle, admits exactly one partition into words from X. It is circular if it is k-circular for every integer k. While it is not a priori clear from the definition, there exists, for every pair [Formula: see text], an integer k such that every k-circular [Formula: see text]-letter code over an alphabet of cardinality n is circular, and we determine the least such integer k for all values of n and [Formula: see text]. The k-circular codes may represent an important evolutionary step between the circular codes, such as the comma-free codes, and the genetic code.

Single-Cell Transcriptomics Reveal a Correlation between Genome Architecture and Gene Family Evolution in Ciliates.

Ciliates, a eukaryotic clade that is over 1 billion years old, are defined by division of genome function between transcriptionally inactive germline micronuclei and functional somatic macronuclei. To date, most analyses of gene family evolution have been limited to cultivable model lineages (e.g., Tetrahymena, Paramecium, Oxytricha, and Stylonychia). Here, we focus on the uncultivable Karyorelictea and its understudied sister class Heterotrichea, which represent two extremes in genome architecture. Somatic macronuclei within the Karyorelictea are described as nearly diploid, while the Heterotrichea have hyperpolyploid somatic genomes. Previous analyses indicate that genome architecture impacts ciliate gene family evolution as the most diverse and largest gene families are found in lineages with extensively processed somatic genomes (i.e., possessing thousands of gene-sized chromosomes). To further assess ciliate gene family evolution, we analyzed 43 single-cell transcriptomes from 33 ciliate species representing 10 classes. Focusing on conserved eukaryotic genes, we use estimates of transcript diversity as a proxy for the number of paralogs in gene families among four focal clades: Karyorelictea, Heterotrichea, extensive fragmenters (with gene-size somatic chromosomes), and non-extensive fragmenters (with more traditional somatic chromosomes), the latter two within the subphylum Intramacronucleata. Our results show that (i) the Karyorelictea have the lowest average transcript diversity, while Heterotrichea are highest among the four groups; (ii) proteins in Karyorelictea are under the highest functional constraints, and the patterns of selection in ciliates may reflect genome architecture; and (iii) stop codon reassignments vary among members of the Heterotrichea and Spirotrichea but are conserved in other classes.IMPORTANCE To further our understanding of genome evolution in eukaryotes, we assess the relationship between patterns of molecular evolution within gene families and variable genome structures found among ciliates. We combine single-cell transcriptomics with bioinformatic tools, focusing on understudied and uncultivable lineages selected from across the ciliate tree of life. Our analyses show that genome architecture correlates with patterns of protein evolution as lineages with more canonical somatic genomes, such as the class Karyorelictea, have more conserved patterns of molecular evolution compared to other classes. This study showcases the power of single-cell transcriptomics for investigating genome architecture and evolution in uncultivable microbial lineages and provides transcriptomic resources for further research on genome evolution.

The Standard Genetic Code can Evolve from a Two-Letter GC Code Without Information Loss or Costly Reassignments.

It is widely agreed that the standard genetic code must have been preceded by a simpler code that encoded fewer amino acids. How this simpler code could have expanded into the standard genetic code is not well understood because most changes to the code are costly. Taking inspiration from the recently synthesized six-letter code, we propose a novel hypothesis: the initial genetic code consisted of only two letters, G and C, and then expanded the number of available codons via the introduction of an additional pair of letters, A and U. Various lines of evidence, including the relative prebiotic abundance of the earliest assigned amino acids, the balance of their hydrophobicity, and the higher GC content in genome coding regions, indicate that the original two nucleotides were indeed G and C. This process of code expansion probably started with the third base, continued with the second base, and ended up as the standard genetic code when the second pair of letters was introduced into the first base. The proposed process is consistent with the available empirical evidence, and it uniquely avoids the problem of costly code changes by positing instead that the code expanded its capacity via the creation of new codons with extra letters.

Frozen Accident Pushing 50: Stereochemistry, Expansion, and Chance in the Evolution of the Genetic Code.

Nearly 50 years ago, Francis Crick propounded the frozen accident scenario for the evolution of the genetic code along with the hypothesis that the early translation system consisted primarily of RNA. Under the frozen accident perspective, the code is universal among modern life forms because any change in codon assignment would be highly deleterious. The frozen accident can be considered the default theory of code evolution because it does not imply any specific interactions between amino acids and the cognate codons or anticodons, or any particular properties of the code. The subsequent 49 years of code studies have elucidated notable features of the standard code, such as high robustness to errors, but failed to develop a compelling explanation for codon assignments. In particular, stereochemical affinity between amino acids and the cognate codons or anticodons does not seem to account for the origin and evolution of the code. Here, I expand Crick's hypothesis on RNA-only translation system by presenting evidence that this early translation already attained high fidelity that allowed protein evolution. I outline an experimentally testable scenario for the evolution of the code that combines a distinct version of the stereochemical hypothesis, in which amino acids are recognized via unique sites in the tertiary structure of proto-tRNAs, rather than by anticodons, expansion of the code via proto-tRNA duplication, and the frozen accident.

An Informational Study of the Evolution of Codes and of Emerging Concepts in Populations of Agents.

We consider the problem of the evolution of a code within a structured population of agents. The agents try to maximize their information about their environment by acquiring information from the outputs of other agents in the population. A naive use of information-theoretic methods would assume that every agent knows how to interpret the information offered by other agents. However, this assumes that it knows which other agents it observes, and thus which code they use. In our model, however, we wish to preclude that: It is not clear which other agents an agent is observing, and the resulting usable information is therefore influenced by the universality of the code used and by which agents an agent is listening to. We further investigate whether an agent that does not directly perceive the environment can distinguish states by observing other agents' outputs. For this purpose, we consider a population of different types of agents talking about different concepts, and try to extract new ones by considering their outputs only.

Quaternionic representation of the genetic code.

A heuristic diagram of the evolution of the standard genetic code is presented. It incorporates, in a way that resembles the energy levels of an atom, the physical notion of broken symmetry and it is consistent with original ideas by Crick on the origin and evolution of the code as well as with the chronological order of appearance of the amino acids along the evolution as inferred from work that mixtures known experimental results with theoretical speculations. Suggested by the diagram we propose a Hamilton quaternions based mathematical representation of the code as it stands now-a-days. The central object in the description is a codon function that assigns to each amino acid an integer quaternion in such a way that the observed code degeneration is preserved. We emphasize the advantages of a quaternionic representation of amino acids taking as an example the folding of proteins. With this aim we propose an algorithm to go from the quaternions sequence to the protein three dimensional structure which can be compared with the corresponding experimental one stored at the Protein Data Bank. In our criterion the mathematical representation of the genetic code in terms of quaternions merits to be taken into account because it describes not only most of the known properties of the genetic code but also opens new perspectives that are mainly derived from the close relationship between quaternions and rotations.

The "periodic table" of the genetic code: A new way to look at the code and the decoding process.

Henri Grosjean and Eric Westhof recently presented an information-rich, alternative view of the genetic code, which takes into account current knowledge of the decoding process, including the complex nature of interactions between mRNA, tRNA and rRNA that take place during protein synthesis on the ribosome, and it also better reflects the evolution of the code. The new asymmetrical circular genetic code has a number of advantages over the traditional codon table and the previous circular diagrams (with a symmetrical/clockwise arrangement of the U, C, A, G bases). Most importantly, all sequence co-variances can be visualized and explained based on the internal logic of the thermodynamics of codon-anticodon interactions.

Mutations enabling displacement of tryptophan by 4-fluorotryptophan as a canonical amino acid of the genetic code.

The 20 canonical amino acids of the genetic code have been invariant over 3 billion years of biological evolution. Although various aminoacyl-tRNA synthetases can charge their cognate tRNAs with amino acid analogs, there has been no known displacement of any canonical amino acid from the code. Experimental departure from this universal protein alphabet comprising the canonical amino acids was first achieved in the mutants of the Bacillus subtilis QB928 strain, which after serial selection and mutagenesis led to the HR23 strain that could use 4-fluorotryptophan (4FTrp) but not canonical tryptophan (Trp) for propagation. To gain insight into this displacement of Trp from the genetic code by 4FTrp, genome sequencing was performed on LC33 (a precursor strain of HR23), HR23, and TR7 (a revertant of HR23 that regained the capacity to propagate on Trp). Compared with QB928, the negative regulator mtrB of Trp transport was found to be knocked out in LC33, HR23, and TR7, and sigma factor sigB was mutated in HR23 and TR7. Moreover, rpoBC encoding RNA polymerase subunits were mutated in three independent isolates of TR7 relative to HR23. Increased expression of sigB was also observed in HR23 and in TR7 growing under 4FTrp. These findings indicated that stabilization of the genetic code can be provided by just a small number of analog-sensitive proteins, forming an oligogenic barrier that safeguards the canonical amino acids throughout biological evolution.

Evidence for late resolution of the aux codon box in evolution.

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNA(Ile) isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNA(Ile) precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNA(Ile) that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNA(Ile) rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNACAU(Met) from near-cognate tRNACAU(Ile). Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNA(Ile) identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades.