Structural Enzymology, Phylogenetics, Differentiation, and Symbolic Reflexivity at the Dawn of Biology.

Unlabelled: The reflexive translation of symbols in one chemical language to another defined genetics. Yet, the co-linearity of codons and amino acids is so commonplace an idea that few even ask how it arose. Readout is done by two distinct sets of proteins, called aminoacyl-tRNA synthetases (AARS).

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes.

The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS 'urzymes'. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes.

AARS Online: A collaborative database on the structure, function, and evolution of the aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRS) are a large group of enzymes that implement the genetic code in all known biological systems. They attach amino acids to their cognate tRNAs, moonlight in various translational and non-translational activities beyond aminoacylation, and are linked to many genetic disorders. The aaRS have a subtle ontology characterized by structural and functional idiosyncrasies that vary from organism to organism, and protein to protein.

HetMM: A Michaelis-Menten model for non-homogeneous enzyme mixtures.

The Michaelis-Menten model requires its reaction velocities to come from a preparation of homogeneous enzymes, with identical or near-identical catalytic activities. However, this condition is not always met. We introduce a kinetic model that relaxes this requirement, by assuming there are an unknown number of enzyme species drawn from a probability distribution whose standard deviation is estimated.

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme.

The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional "urzymes" from TrpRS, LeuRS, and HisRS.

Enzymic recognition of amino acids drove the evolution of primordial genetic codes.

How genetic information gained its exquisite control over chemical processes needed to build living cells remains an enigma. Today, the aminoacyl-tRNA synthetases (AARS) execute the genetic codes in all living systems. But how did the AARS that emerged over three billion years ago as low-specificity, protozymic forms then spawn the full range of highly-specific enzymes that distinguish between 22 diverse amino acids? A phylogenetic reconstruction of extant AARS genes, enhanced by analysing modular acquisitions, reveals six AARS with distinct bacterial, archaeal, eukaryotic, or organellar clades, resulting in a total of 36 families of AARS catalytic domains.

Origins of Genetic Coding: Self-Guided Molecular Self-Organisation.

The origin of genetic coding is characterised as an event of cosmic significance in which quantum mechanical causation was transcended by constructive computation. Computational causation entered the physico-chemical processes of the pre-biotic world by the incidental satisfaction of a condition of reflexivity between polymer sequence information and system elements able to facilitate their own production through translation of that information. This event, which has previously been modelled in the dynamics of Gene-Replication-Translation systems, is properly described as a process of self-guided self-organisation.

Multidimensional Phylogenetic Metrics Identify Class I Aminoacyl-tRNA Synthetase Evolutionary Mosaicity and Inter-Modular Coupling.

The role of aminoacyl-tRNA synthetases (aaRS) in the emergence and evolution of genetic coding poses challenging questions concerning their provenance. We seek evidence about their ancestry from curated structure-based multiple sequence alignments of a structurally invariant "scaffold" shared by all 10 canonical Class I aaRS. Three uncorrelated phylogenetic metrics-mutation frequency, its uniformity, and row-by-row cladistic congruence-imply that the Class I scaffold is a mosaic assembled from successive genetic sources.

The Roots of Genetic Coding in Aminoacyl-tRNA Synthetase Duality.

Codon-dependent translation underlies genetics and phylogenetic inferences, but its origins pose two challenges. Prevailing narratives cannot account for the fact that aminoacyl-tRNA synthetases (aaRSs), which translate the genetic code, must collectively enforce the rules used to assemble themselves. Nor can they explain how specific assignments arose from rudimentary differentiation between ancestral aaRSs and corresponding transfer RNAs (tRNAs).

Reciprocally-Coupled Gating: Strange Loops in Bioenergetics, Genetics, and Catalysis.

Bioenergetics, genetic coding, and catalysis are all difficult to imagine emerging without pre-existing historical context. That context is often posed as a "Chicken and Egg" problem; its resolution is concisely described by de Grasse Tyson: "The egg was laid by a bird that was not a chicken". The concision and generality of that answer furnish no details-only an appropriate framework from which to examine detailed paradigms that might illuminate paradoxes underlying these three life-defining biomolecular processes.

Impedance Matching and the Choice Between Alternative Pathways for the Origin of Genetic Coding.

We recently observed that errors in gene replication and translation could be seen qualitatively to behave analogously to the impedances in acoustical and electronic energy transducing systems. We develop here quantitative relationships necessary to confirm that analogy and to place it into the context of the minimization of dissipative losses of both chemical free energy and information. The formal developments include expressions for the information transferred from a template to a new polymer, I; an impedance parameter, Z; and an effective alphabet size, n; all of which have non-linear dependences on the fidelity parameter, q, and the alphabet size, n.

Author Correction: Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Ancient Operational Code is Embedded in the Amino Acid Substitution Matrix and aaRS Phylogenies.

The underlying structure of the canonical amino acid substitution matrix (aaSM) is examined by considering stepwise improvements in the differential recognition of amino acids according to their chemical properties during the branching history of the two aminoacyl-tRNA synthetase (aaRS) superfamilies. The evolutionary expansion of the genetic code is described by a simple parameterization of the aaSM, in which (i) the number of distinguishable amino acid types, (ii) the matrix dimension and (iii) the number of parameters, each increases by one for each bifurcation in an aaRS phylogeny. Parameterized matrices corresponding to trees in which the size of an amino acid sidechain is the only discernible property behind its categorization as a substrate, exclusively for a Class I or II aaRS, provide a significantly better fit to empirically determined aaSM than trees with random bifurcation patterns.

Reflexivity, coding and quantum biology.

Biological systems are fundamentally computational in that they process information in an apparently purposeful fashion rather than just transferring bits of it in a purely syntactical manner. Biological information, such has genetic information stored in DNA sequences, has semantic content. It carries meaning that is defined by the molecular context of its cellular environment.

Class I and II aminoacyl-tRNA synthetase tRNA groove discrimination created the first synthetase-tRNA cognate pairs and was therefore essential to the origin of genetic coding.

The genetic code likely arose when a bidirectional gene replicating as a quasi-species began to produce ancestral aminoacyl-tRNA synthetases (aaRS) capable of distinguishing between two distinct sets of amino acids. The synthetase class division therefore necessarily implies a mechanism by which the two ancestral synthetases could also discriminate between two different kinds of tRNA substrates. We used regression methods to uncover the possible patterns of base sequences capable of such discrimination and find that they appear to be related to thermodynamic differences in the relative stabilities of a hairpin necessary for recognition of tRNA substrates by Class I aaRS.

Experimental solutions to problems defining the origin of codon-directed protein synthesis.

How genetic coding differentiated biology from chemistry is a long-standing challenge in Biology, for which there have been few experimental approaches, despite a wide-ranging speculative literature. We summarize five coordinated areas-experimental characterization of functional approximations to the minimal peptides (protozymes and urzymes) necessary to activate amino acids and acylate tRNA; showing that specificities of these experimental models match those expected from the synthetase Class division; population of disjoint regions of amino acid sequence space via bidirectional coding ancestry of the two synthetase Classes; showing that the phase transfer equilibria of amino acid side chains that form a two-dimensional basis set for protein folding are embedded in patterns of bases in the tRNA acceptor stem and anticodon; and identification of molecular signatures of ancestral synthetases and tRNAs necessary to define the earliest cognate synthetase:tRNA pairs-that now compose an extensive experimentally testable paradigm for progress toward understanding the coordinated emergence of the codon table and viable mRNA coding sequences. We briefly discuss recent progress toward identifying the remaining outstanding questions-the nature of the earliest amino acid alphabets and the origin of binding discrimination via distinct amino acid sequence-independent protein secondary structures-and how these, too, might be addressed experimentally.

Quantitative interpretation of isopiestic measurements on aqueous solutions: Urea revisited.

This investigation amends the analysis of isopiestic measurements of solvent thermodynamic activity by taking into account the fact that the solvent activity, traditionally expressed in mole-fraction terms, is a molal parameter because of the constraints (constant temperature and pressure) under which the measurements are made. Application of the revised procedure to published isopiestic measurements on aqueous urea solutions at 25 °C yields a dimerization constant of 0.066 molal, which is two-fold larger than an earlier published estimate based on an incorrect definition of the solute activity coefficient.

Octa-repeat domain of the mammalian prion protein mRNA forms stable A-helical hairpin structure rather than G-quadruplexes.

Misfolding and aggregation of prion protein (PrP) causes neurodegenerative diseases like Creutzfeldt-Jakob disease (CJD) and scrapie. Besides the consensus that spontaneous conversion of normal cellular PrP into misfolded and aggregating PrP is the central event in prion disease, an alternative hypothesis suggests the generation of pathological PrP by rare translational frameshifting events in the octa-repeat domain of the PrP mRNA. Ribosomal frameshifting most commonly relies on a slippery site and an adjacent stable RNA structure to stall translating ribosome.

Hierarchical groove discrimination by Class I and II aminoacyl-tRNA synthetases reveals a palimpsest of the operational RNA code in the tRNA acceptor-stem bases.

Class I and II aaRS recognition of opposite grooves was likely among the earliest determinants fixed in the tRNA acceptor stem bases. A new regression model identifies those determinants in bacterial tRNAs. Integral coefficients relate digital dependent to independent variables with perfect agreement between observed and calculated grooves for all twenty isoaccepting tRNAs.

Life’s most distinguishing feature: meaningful information processing.

The origin of life out of molecular disorder represents an extraordinary transition in the local fabric of the cosmos. The result can only be described by language that is imbued with echoes of purpose and agency. The «codescript» information stored in genes cannot be adequately understood in terms of Shannon’s syntactical measure.

Interdependence, Reflexivity, Fidelity, Impedance Matching, and the Evolution of Genetic Coding.

Genetic coding is generally thought to have required ribozymes whose functions were taken over by polypeptide aminoacyl-tRNA synthetases (aaRS). Two discoveries about aaRS and their interactions with tRNA substrates now furnish a unifying rationale for the opposite conclusion: that the key processes of the Central Dogma of molecular biology emerged simultaneously and naturally from simple origins in a peptide•RNA partnership, eliminating the epistemological utility of a prior RNA world. First, the two aaRS classes likely arose from opposite strands of the same ancestral gene, implying a simple genetic alphabet.

Insuperable problems of the genetic code initially emerging in an RNA world.

Differential equations for error-prone information transfer (template replication, transcription or translation) are developed in order to consider, within the theory of autocatalysis, the advent of coded protein synthesis. Variations of these equations furnish a basis for comparing the plausibility of contrasting scenarios for the emergence of specific tRNA aminoacylation, ultimately by enzymes, and the relationship of this process with the origin of the universal system of molecular biological information processing embodied in the Central Dogma. The hypothetical RNA World does not furnish an adequate basis for explaining how this system came into being, but principles of self-organisation that transcend Darwinian natural selection furnish an unexpectedly robust basis for a rapid, concerted transition to genetic coding from a peptide·RNA world.

Rigorous analysis of static light scattering measurements on buffered protein solutions.

Attention is drawn to the thermodynamic invalidity of the current practice of analyzing static light scattering measurements on globular proteins in terms of theory for a single solute because of its disregard of the need to consider small species such as buffer components as additional cosolutes rather than as part of the solvent. This practice continues despite its demonstrated inadequacy in studies of sucrose-supplemented protein solutions, where the aberrant behavior was recognized to be a consequence of physical protein interaction with the small cosolute. Failure to take into account the consequences of small cosolute effects renders extremely difficult any attempt to obtain a rigorous thermodynamic characterization of protein interactions by this empirical technique.

A Hilly path through the thermodynamics and statistical mechanics of protein solutions.

The opus of Don Winzor in the fields of physical and analytical biochemistry is a major component of that certain antipodean approach to this broad area of research that blossomed in the second half of the twentieth century. The need to formulate problems in terms of thermodynamic nonideality posed the challenge of describing a clear route from molecular interactions to the parameters that biochemists routinely measure. Mapping out this route required delving into the statistical mechanics of solutions of macromolecules, and at every turn mathematically complex, rigorous, general results that had previously been derived previously, often by Terrell Hill, came to the fore.