Construction of genetic code from evolutionary stability

The construction of the genetic code is investigated based on a stability principle. The concept and formulation of mutational deterioration (MD) of the genetic code is proposed. It is proved that the degeneracies of codon multiplets obey the rule to best resist MD. The MD for each ideal multiplet of codons is expressed by four parameters and it takes on a minimum value for real distributions of codons in the multiplet. Then the global mutational deterioration (GMD) of code table is calculated and the minimal code is deduced. The domain-like distribution of hydrophobic and hydrophilic amino acids on the genetic code is explained from the minimization of GMD. It is demonstrated that the standard code is approximately GMD-minimal. By introducing some constraints that are related to the initial condition of the system, we have deduced the standard genetic code from the minimization of GMD. The minimization shows the general trend of evolutionary process to some stable state while the constraints reflect a 'frozen accident.' Many deviant codon assignments are also explained through MD minimization assuming the changeable degrees of degeneracies for some multiplets. So, a possible answer to the question of "Why are synonymous codons and amino acids distributed in the code table just as they are?" is given.     

Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function

Fifty years have passed since the genetic code was deciphered, but how the genetic code came into being has not been satisfactorily addressed. It is now widely accepted that the earliest genetic code did not encode all 20 amino acids found in the universal genetic code as some amino acids have complex biosynthetic pathways and likely were not available from the environment. Therefore, the genetic code evolved as pathways for synthesis of new amino acids became available. One hypothesis proposes that early in the evolution of the genetic code four amino acids—valine, alanine, aspartic acid, and glycine—were coded by GNC codons (N = any base) with the remaining codons being nonsense codons. The other sixteen amino acids were subsequently added to the genetic code by changing nonsense codons into sense codons for these amino acids. Improvement in protein function is presumed to be the driving force behind the evolution of the code, but how improved function was achieved by adding amino acids has not been examined. Based on an analysis of amino acid function in proteins, an evolutionary mechanism for expansion of the genetic code is described in which individual coded amino acids were replaced by new amino acids that used nonsense codons differing by one base change from the sense codons previously used. The improved or altered protein function afforded by the changes in amino acid function provided the selective advantage underlying the expansion of the genetic code. Analysis of amino acid properties and functions explains why amino acids are found in their respective positions in the genetic code.     

A Realistic Model Under Which the Genetic Code is Optimal

The genetic code has a high level of error robustness. Using values of hydrophobicity scales as a proxy for amino acid character, and the mean square measure as a function quantifying error robustness, a value can be obtained for a genetic code which reflects the error robustness of that code. By comparing this value with a distribution of values belonging to codes generated by random permutations of amino acid assignments, the level of error robustness of a genetic code can be quantified. We present a calculation in which the standard genetic code is shown to be optimal. We obtain this result by (1) using recently updated values of polar requirement as input; (2) fixing seven assignments (Ile, Trp, His, Phe, Tyr, Arg, and Leu) based on aptamer considerations; and (3) using known biosynthetic relations of the 20 amino acids. This last point is reflected in an approach of subdivision (restricting the random reallocation of assignments to amino acid subgroups, the set of 20 being divided in four such subgroups). The three approaches to explain robustness of the code (specific selection for robustness, amino acid–RNA interactions leading to assignments, or a slow growth process of assignment patterns) are reexamined in light of our findings. We offer a comprehensive hypothesis, stressing the importance of biosynthetic relations, with the code evolving from an early stage with just glycine and alanine, via intermediate stages, towards 64 codons carrying todays meaning.     

Metabolic Basis for the Self-Referential Genetic Code

An investigation of the biosynthesis pathways producing glycine and serine was necessary to clarify an apparent inconsistency between the self-referential model (SRM) for the formation of the genetic code and the model of coevolution of encodings and of amino acid biosynthesis routes. According to the SRM proposal, glycine was the first amino acid encoded, followed by serine. The coevolution model does not state precisely which the first encodings were, only presenting a list of about ten early assignments including the derivation of glycine from serine—this being derived from the glycolysis intermediate glycerate, which reverses the order proposed by the self-referential model. Our search identified the glycine-serine pathway of syntheses based on one-carbon sources, involving activities of the glycine decarboxylase complex and its associated serine hydroxymethyltransferase, which is consistent with the order proposed by the self-referential model and supports its rationale for the origin of the genetic code: protein synthesis was developed inside an early metabolic system, serving the function of a sink of amino acids; the first peptides were glycine-rich and fit for the function of building the early ribonucleoproteins; glycine consumption in proteins drove the fixation of the glycine-serine pathway.     

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     

Engineering Translation Components for Genetic Code Expansion

The expansion of the genetic code consisting of four bases and 20 amino acids into diverse building blocks has been an exciting topic in synthetic biology. Many biochemical components are involved in gene expression; therefore, adding a new component to the genetic code requires engineering many other components that interact with it. Genetic code expansion has advanced significantly for the last two decades with the engineering of several components involved in protein synthesis. These components include tRNA/aminoacyl-tRNA synthetase, new codons, ribosomes, and elongation factor Tu. In addition, biosynthesis and enhanced uptake of non-canonical amino acids have been attempted and have made meaningful progress. This review discusses the efforts to engineer these translation components, to improve the genetic code expansion technology.     

Genetic Code Expansion Through Quadruplet Codon Decoding

Noncanonical amino acid mutagenesis has emerged as a powerful tool for the study of protein structure and function. While triplet nonsense codons, especially the amber codon, have been widely employed, quadruplet codons have attracted attention for the potential of creating additional blank codons for noncanonical amino acids mutagenesis. In this review, we discuss methodologies and applications of quadruplet codon decoding in genetic code expansion both in vitro and in vivo.     

Codon Usage Decreases the Error Minimization Within the Genetic Code

The genetic code is not random but instead is organized in such a way that single nucleotide substitutions are more likely to result in changes between similar amino acids. This fidelity, or error minimization, has been proposed to be an adaptation within the genetic code. Many models have been proposed to measure this adaptation within the genetic code. However, we find that none of these consider codon usage differences between species. Furthermore, use of different indices of amino acid physicochemical characteristics leads to different estimations of this adaptation within the code. In this study, we try to establish a more accurate model to address this problem. In our model, a weighting scheme is established for mistranslation biases of the three different codon positions, transition/transversion biases, and codon usage. Different indices of amino acids physicochemical characteristics are also considered. In contrast to pervious work, our results show that the natural genetic code is not fully optimized for error minimization. The genetic code, therefore, is not the most optimized one for error minimization, but one that balances between flexibility and fidelity for different species.     

The Level and Landscape of Optimization in the Origin of the Genetic Code

We consider a model of the origin of genetic code organization incorporating the biosynthetic relationships between amino acids and their physicochemical properties. We study the behavior of the genetic code in the set of codes subject both to biosynthetic constraints and to the constraint that the biosynthetic classes of amino acids must occupy only their own codon domain, as observed in the genetic code. Therefore, this set contains the smallest number of elements ever analyzed in similar studies. Under these conditions and if, as predicted by physicochemical postulates, the amino acid properties played a fundamental role in genetic code organization, it can be expected that the code must display an extremely high level of optimization. This prediction is not supported by our analysis, which indicates, for instance, a minimization percentage of only 80%. These observations can therefore be more easily explained by the coevolution theory of genetic code origin, which postulates a role that is important but not fundamental for the amino acid properties in the structuring of the code. We have also investigated the shape of the optimization landscape that might have arisen during genetic code origin. Here, too, the results seem to favor the coevolution theory because, for instance, the fact that only a few amino acid exchanges would have been sufficient to transform the genetic code (which is not a local minimum) into a much better optimized code, and that such exchanges did not actually take place, seems to suggest that, for instance, the reduction of translation errors was not the main adaptive theme structuring the genetic code.     

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     

Signature of a Primitive Genetic Code in Ancient Protein Lineages

The genetic code is the syntactic foundation underlying the structure and function of every protein in the history of the biological world. Its highly ordered degenerate complexity suggests an incremental evolution, the result of a combination of selective, mechanistic, and random processes. These evolutionary processes are still poorly understood and remain an open question in the study of early life on Earth. We perform a compositional analysis of ribosomal proteins and ATPase subunits in bacterial and archaeal lineages, using conserved positions that came and remained under purifying selection before and up to the most recent common ancestor. An observable shift in amino acid usage at these conserved positions likely provides an untapped window into the history of protein sequence space, allowing events of genetic code expansion to be identified. We identify Cys, Glu, Phe, Ile, Lys, Val, Trp, and Tyr as recent additions to the genetic code, with Asn, Gln, Gly, and Leu among the more ancient. Our observations are consistent with a scenario in which genetic code expansion primarily favored amino acids that promoted an increase in polypeptide size and functionality. We propose that this expansion would have been critical in the takeover of many RNA-mediated processes, as well as the addition of novel biological functions inaccessible to an RNA-based physiology, such as crossing lipid membranes. Thus, expansion of the genetic code likely set the stage for the transition from RNA-based to protein-based life. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Little Evidence the Standard Genetic Code Is Optimized for Resource Conservation

Selection for resource conservation can shape the coding sequences of organisms living in nutrient-limited environments. Recently, it was proposed that selection for resource conservation, specifically for nitrogen and carbon content, has also shaped the structure of the standard genetic code, such that the missense mutations the code allows tend to cause small increases in the number of nitrogen and carbon atoms in amino acids. Moreover, it was proposed that this optimization is not confounded by known optimizations of the standard genetic code, such as for polar requirement or hydropathy. We challenge these claims. We show the proposed optimization for nitrogen conservation is highly sensitive to choice of null model and the proposed optimization for carbon conservation is confounded by the known conservative nature of the standard genetic code with respect to the molecular volume of amino acids. There is therefore little evidence the standard genetic code is optimized for resource conservation. We discuss our findings in the context of null models of the standard genetic code.     

The distribution of amino acids in the genetic code

By introducing a mutational deterioration functionMD and a principle of approximate minimum of the function, we have deduced the distribution of amino acids in genetic code, which includes the degenracy rule of codons, the global extreme of genetic code from codon interactions and the hydrophobicity domain of the prevalent (standard) code.The project supported by National Science Foundation of China.     

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.     

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.     

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.     

A Novel Theory on the Origin of the Genetic Code: A GNC-SNS Hypothesis

We have previously proposed an SNS hypothesis on the origin of the genetic code (Ikehara and Yoshida 1998). The hypothesis predicts that the universal genetic code originated from the SNS code composed of 16 codons and 10 amino acids (S and N mean G or C and either of four bases, respectively). But, it must have been very difficult to create the SNS code at one stroke in the beginning. Therefore, we searched for a simpler code than the SNS code, which could still encode water-soluble globular proteins with appropriate three-dimensional structures at a high probability using four conditions for globular protein formation (hydropathy, α-helix, β-sheet, and β-turn formations). Four amino acids (Gly [G], Ala [A], Asp [D], and Val [V]) encoded by the GNC code satisfied the four structural conditions well, but other codes in rows and columns in the universal genetic code table do not, except for the GNG code, a slightly modified form of the GNC code. Three three-amino acid systems ([D], Leu and Tyr; [D], Tyr and Met; Glu, Pro and Ile) also satisfied the above four conditions. But, some amino acids in the three systems are far more complex than those encoded by the GNC code. In addition, the amino acids in the three-amino acid systems are scattered in the universal genetic code table. Thus, we concluded that the universal genetic code originated not from a three-amino acid system but from a four-amino acid system, the GNC code encoding [GADV]-proteins, as the most primitive genetic code. Received: 11 June 2001 / Accepted: 11 October 2001     

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.