On Error Minimization in a Sequential Origin of the Standard Genetic Code

Distances between amino acids were derived from the polar requirement measure of amino acid polarity and Benner and co-workers' (1994) 74-100 PAM matrix. These distances were used to examine the average effects of amino acid substitutions due to single-base errors in the standard genetic code and equally degenerate randomized variants of the standard code. Second-position transitions conserved all distances on average, an order of magnitude more than did second-position transversions. In contrast, first-position transitions and transversions were about equally conservative. In comparison with randomized codes, second-position transitions in the standard code significantly conserved mean square differences in polar requirement and mean Benner matrix-based distances, but mean absolute value differences in polar requirement were not significantly conserved. The discrepancy suggests that these commonly used distance measures may be insufficient for strict hypothesis testing without more information. The translational consequences of single-base errors were then examined in different codon contexts, and similarities between these contexts explored with a hierarchical cluster analysis. In one cluster of codon contexts corresponding to the RNY and GNR codons, second-position transversions between C and G and transitions between C and U were most conservative of both polar requirement and the matrix-based distance. In another cluster of codon contexts, second-position transitions between A and G were most conservative. Despite the claims of previous authors to the contrary, it is shown theoretically that the standard code may have been shaped by position-invariant forces such as mutation and base content. These forces may have left heterogeneous signatures in the code because of differences in translational fidelity by codon position. A scenario for the origin of the code is presented wherein selection for error minimization could have occurred multiple times in disjoint parts of the code through a phyletic process of competition between lineages. This process permits error minimization without the disruption of previously useful messages, and does not predict that the code is optimally error-minimizing with respect to modern error. Instead, the code may be a record of genetic process and patterns of mutation before the radiation of modern organisms and organelles. Received: 28 July 1997 / Accepted: 23 January 1998     

The Case for an Error Minimizing Standard Genetic Code

Since discovering the pattern by which amino acids are assigned to codons within the standard genetic code, investigators have explored the idea that natural selection placed biochemically similar amino acids near to one another in coding space so as to minimize the impact of mutations and/or mistranslations. The analytical evidence to support this theory has grown in sophistication and strength over the years, and counterclaims questioning its plausibility and quantitative support have yet to transcend some significant weaknesses in their approach. These weaknesses are illustrated here by means of a simple simulation model for adaptive genetic code evolution. There remain ill explored facets of the `error minimizing' code hypothesis, however, including the mechanism and pathway by which an adaptive pattern of codon assignments emerged, the extent to which natural selection created synonym redundancy, its role in shaping the amino acid and nucleotide languages, and even the correct interpretation of the adaptive codon assignment pattern: these represent fertile areas for future research.     

Coding Rules for Amino Acids in the Genetic Code: The Genetic Code is a Minimal Code of Mutational Deterioration

Coding rules for amino acids in the genetic code are discussed from the point that the genetic code is a minimal code ofmutational deterioration. The global mutational deterioration(GMD) function is defined through several parameters describingsingle base mutations and amino acid distances. The problem ofsearching for the global minimum of the GMD function is discussedin some detail. From GMD minimization under initial constraintswe have succeeded in deducing the standard genetic code.     

Factors in Protobiomonomer Selection for the Origin of the Standard Genetic Code

Acta Biotheoretica - Natural selection of specific protobiomonomers during abiogenic development of the prototype genetic code is hindered by the diversity of structural, spatial, and rotational...     

Applications of Genetic Code Expansion in Studying Protein Post-translational Modification

Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and sub-stoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation/nitration/hydroxylation, Lys acetylation/acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described.     

Pathways of Genetic Adaptation: Multistep Origin of Mutants Under Selection Without Induced Mutagenesis in Salmonella enterica

In several bacterial systems, mutant cell populations plated on growth-restricting medium give rise to revertant colonies that accumulate over several days. One model suggests that nongrowing parent cells mutagenize their own genome and thereby create beneficial mutations (stress-induced mutagenesis). By this model, the first-order induction of new mutations in a nongrowing parent cell population leads to the delayed accumulation of visible colonies. In an alternative model (selection only), selective conditions allow preexisting small-effect mutants to initiate clones that grow and give rise to faster-growing mutants. By the selection-only model, the delay in appearance of revertant colonies reflects (1) the time required for initial clones to reach a size sufficient to allow the second mutation plus (2) the time required for growth of the improved subclone. We previously characterized a system in which revertant colonies accumulate slowly and contain cells with two mutations, one formed before plating and one after. This left open the question of whether mutation rates increase under selection. Here we measure the unselected formation rate and the growth contribution of each mutant type. When these parameters are used in a graphic model of revertant colony development, they demonstrate that no increase in mutation rate is required to explain the number and delayed appearance of two of the revertant types.     

An Analysis of the Metabolic Theory of the Origin of the Genetic Code

A computer program was used to test Wong's coevolution theory of the genetic code. The codon correlations between the codons of biosynthetically related amino acids in the universal genetic code and in randomly generated genetic codes were compared. It was determined that many codon correlations are also present within random genetic codes and that among the random codes there are always several which have many more correlations than that found in the universal code. Although the number of correlations depends on the choice of biosynthetically related amino acids, the probability of choosing a random genetic code with the same or greater number of codon correlations as the universal genetic code was found to vary from 0.1% to 34% (with respect to a fairly complete listing of related amino acids). Thus, Wong's theory that the genetic code arose by coevolution with the biosynthetic pathways of amino acids, based on codon correlations between biosynthetically related amino acids, is statistical in nature. Received: 8 August 1996 / Accepted: 26 December 1996     

On the Structural Regularity in Nucleobases and Amino Acids and Relationship to the Origin and Evolution of the Genetic Code

To explore how chemical structures of both nucleobases and amino acids may have played a role in shaping the genetic code, numbers of sp² hybrid nitrogen atoms in nucleobases were taken as a determinative measure for empirical stereo-electronic property to analyze the genetic code. Results revealed that amino acid hydropathy correlates strongly with the sp² nitrogen atom numbers in nucleobases rather than with the overall electronic property such as redox potentials of the bases, reflecting that stereo-electronic property of bases may play a role. In the rearranged code, five simple but stereo-structurally distinctive amino acids (Gly, Pro, Val, Thr and Ala) and their codon quartets form a crossed intersection “core”. Secondly, a re-categorization of the amino acids according to their β-carbon stereochemistry, verified by charge density (at β-carbon) calculation, results in five groups of stereo-structurally distinctive amino acids, the group leaders of which are Gly, Pro, Val, Thr and Ala, remarkably overlapping the above “core”. These two lines of independent observations provide empirical arguments for a contention that a seemingly “frozen” “core” could have formed at a certain evolutionary stage. The possible existence of this codon “core” is in conformity with a previous evolutionary model whereby stereochemical interactions may have shaped the code. Moreover, the genetic code listed in UCGA succession together with this codon “core” has recently facilitated an identification of the unprecedented icosikaioctagon symmetry and bi-pyramidal nature of the genetic code.     

The Robust Statistical Bases of the Coevolution Theory of Genetic Code Origin

A paper (Amirnovin R, J Mol Evol 44:473–476, 1997) seems to undermine the validity of the coevolution theory of genetic code origin by shedding doubt on the connection between the biosynthetic relationships between amino acids and the organization of the genetic code, at a time when the literature on the topic takes this for granted. However, as a few papers cite this paper as evidence against the coevolution theory, and to cast aside all doubt on the subject, we have decided to reanalyze the statistical bases on which this theory is founded. We come to the following conclusions: (1) the methods used in the above referred paper contain certain mistakes, and (2) the statistical foundations on which the coevolution theory is based are extremely robust. We have done this by critically appraising Amirnovin's paper and suggesting an alternative method based on the generation of random codes which, along with the method reported in the literature, allows us to evaluate the significance, in the genetic code, of different sets of amino acid pairs in biosynthetic relationships. In particular, by using this method and after building up a certain set of amino acid pairs reflecting the expectations of the coevolution theory, we show that the presence of this set in the genetic code would be obtained, purely by chance, with a probability of 6 × 10⁻⁵. This observation seems to provide particularly strong support to the coevolution theory. Received: 28 June 1999 / Accepted: 23 October 1999     

A Blind Empiricism Against the Coevolution Theory of the Origin of the Genetic Code

Ronneberg et al. (Proc Natl Acad Sci USA 97:13690–13695, 2000) recently suggested abandoning the coevolution theory of genetic code origin on the basis of two pieces of evidence. They (1) criticize the use of several pairs of amino acids in a precursor–product relationship to support this theory and (2) suggest a new set of codes in which to investigate the statistical bases of the coevolution theory, reaching the conclusion that this theory is not statistically validated in this set. In this paper I critically analyze the robustness of these conclusions. Observations and arguments lead to the belief that the pairs of amino acids in a precursor–product relationship originally used by the coevolution theory are such, or may at least be interpreted as such, and are therefore a manifestation of this theory. Furthermore, the new set of codes that Ronneberg et al. suggest is open to criticism and is thus substituted by the set of amino acid permutation codes, in which even the pairs of amino acids they favor end up by supporting the coevolution theory. Overall, the analysis seems to show that the paper by Ronneberg et al. is of minor scientific value while the coevolution theory seems to be one of the best theories at our disposal for explaining the evolutionary organisation of the genetic code and is, contrary to their claims, statistically well validated. Received: 21 February 2001 / Accepted: 22 May 2001     

Error-reducing Structure of the Genetic Code Indicates Code Origin in Non-thermophile Organisms

During the RNA World, organisms experienced high rates of genetic errors, which implies that there was strong evolutionary pressure to reduce the errors’ phenotypical impact by suitably structuring the still-evolving genetic code. Therefore, the relative rates of the various types of genetic errors should have left characteristic imprints in the structure of the genetic code. Here, we show that, therefore, it is possible to some extent to reconstruct those error rates, as well as the nucleotide frequencies, for the time when the code was fixed. We find evidence indicating that the frequencies of G and C in the genome were not elevated. Since, for thermodynamic reasons, RNA in thermophiles tends to possess elevated G+C content, this result indicates that the fixation of the genetic code occurred in organisms which were either not thermophiles or that the code’s fixation occurred after the rise of DNA. Supplementary Materials Original data and programs are available at the author’s web site: .     

On the Classes of Aminoacyl-tRNA Synthetases,Amino Acids and the Genetic Code

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering models for the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporation of new amino acids following duplication and divergence of previous synthetases and tRNA molecules.     

Physicochemical Optimization in the Genetic Code Origin as the Number of Codified Amino Acids Increases

We have assumed that the coevolution theory of genetic code origin (Wong JT, Proc Natl Acad Sci USA 72:1909–1912, 1975) is essentially correct. This theory makes it possible to identify at least 10 evolutionary stages through which genetic code organization might have passed prior to reaching its current form. The calculation of the minimization level of all these evolutionary stages leads to the following conclusions. (1) The minimization percentages increased linearly with the number of amino acids codified in the codes of the various evolutionary stages when only the sense changes are considered in the analysis. This seems to favor the physicochemical theory of genetic code origin even if, as discussed in the paper, this observation is also compatible with the coevolution theory. (2) For the first seven evolutionary stages of the genetic code, this trend is less clear and indeed is inverted when we consider the global optimisation of the codes due to both sense changes and synonymous changes. This inverse correlation between minimization percentages and the number of amino acids codified in the codes of the intermediate stages seems to favor neither the physicochemical nor the stereochemical theories of genetic code origin, as it is in the early and intermediate stages of code development that these theories would expect minimization to have played a crucial role, and this does not seem to be the case. However, these results are in agreement with the coevolution theory, which attributes a role to the physicochemical properties of amino acids that, while important, is nevertheless subordinate to the mechanism which concedes codons from the precursor amino acids to the product amino acids as the primary factor determining the evolutionary structuring of the genetic code. The results are therefore discussed in the context of the various theories proposed to explain genetic code origin. Received: 25 October 1998 / Accepted: 19 February 1999     

Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX.     

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.     

Recent Technologies for Genetic Code Expansion and their Implications on Synthetic Biology Applications

Genetic code expansion (GCE) enables the site-specific incorporation of non-canonical amino acids as novel building blocks for the investigation and manipulation of proteins. The advancement of genetic code expansion has been benefited from the development of synthetic biology, while genetic code expansion also helps to create more synthetic biology tools. In this review, we summarize recent advances in genetic code expansion brought by synthetic biology progresses, including engineering of the translation machinery, genome-wide codon reassignment, and the biosynthesis of non-canonical amino acids. We highlight the emerging application of this technology in construction of new synthetic biology parts, circuits, chassis, and products.     

On Darwin's palaeontology in The Origin of Species

I investigate the role of palaeontology within Darwin's works through an analysis of the two chapters of The Origin of Species most especially devoted to this science. Palaeontology may occupy several places within the structure of the argumentative logic of Darwinism, but these places have remained to some extent ancillary. Indeed, palaeontology could well document evolutionary patterns, showing the actual occurrence of evolution as a general “historical fact”, but it was poorly adapted to demonstrate the main point of Darwinism: the actual evolutionary process: natural selection acting among individuals. I also show, in agreement with Gould, that Darwin had great confidence in the ultimate ability of palaeontology to support his theory, and that in interpreting palaeontological evidence, he expressed a vision of natural selection much wider and more eclectic than that which has generally been ascribed to him.