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.     

Enzymes with noncanonical amino acids

Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years.     

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     

Some theoretical aspects of reprogramming the standard genetic code

Reprogramming of the standard genetic code to include non-canonical amino acids (ncAAs) opens new prospects for medicine, industry, and biotechnology. There are several methods of code engineering, which allow us for storing new genetic information in DNA sequences and producing proteins with new properties. Here, we provided a theoretical background for the optimal genetic code expansion, which may find application in the experimental design of the genetic code. We assumed that the expanded genetic code includes both canonical and non-canonical information stored in 64 classical codons. What is more, the new coding system is robust to point mutations and minimizes the possibility of reversion from the new to old information. In order to find such codes, we applied graph theory to analyze the properties of optimal codon sets. We presented the formal procedure in finding the optimal codes with various number of vacant codons that could be assigned to new amino acids. Finally, we discussed the optimal number of the newly incorporated ncAAs and also the optimal size of codon groups that can be assigned to ncAAs.     

Asymmetry in the symmetric structure of the genetic code]

The genetic code is characterized by hidden symmetry. Amino acids possessing common antiamino acids are located symmetrically in the graphic models of the code. There is only one exception--apolar amino acids V, M, I, L and F are asymmetrically arranged. Asymmetric disposition of these amino acids is apparently due to divergence in the course of structural evolution of amino acid families as a result of inclusion of new members into the coding system.     

Stability of the genetic code and optimal parameters of amino acids

The standard genetic code is known to be much more efficient in minimizing adverse effects of misreading errors and one-point mutations in comparison with a random code having the same structure, i.e. the same number of codons coding for each particular amino acid. We study the inverse problem, how the code structure affects the optimal physico-chemical parameters of amino acids ensuring the highest stability of the genetic code. It is shown that the choice of two or more amino acids with given properties determines unambiguously all the others. In this sense the code structure determines strictly the optimal parameters of amino acids or the corresponding scales may be derived directly from the genetic code. In the code with the structure of the standard genetic code the resulting values for hydrophobicity obtained in the scheme “leave one out” and in the scheme with fixed maximum and minimum parameters correlate significantly with the natural scale. The comparison of the optimal and natural parameters allows assessing relative impact of physico-chemical and error-minimization factors during evolution of the genetic code. As the resulting optimal scale depends on the choice of amino acids with given parameters, the technique can also be applied to testing various scenarios of the code evolution with increasing number of codified amino acids. Our results indicate the co-evolution of the genetic code and physico-chemical properties of recruited amino acids.     

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.     

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 the 28-gon symmetry inherent in the genetic code intertwined with aminoacyl-tRNA synthetases—The Lucas series

Despite considerable efforts it has remained unclear what principle governs the selection of the 20 canonical amino acids in the genetic code. Based on a previous study of the 28-gonal and rotational symmetric arrangement of the 20 amino acids in the genetic code, new analyses of the organization of the genetic code system together with their intrinsic relation to the two classes of aminoacyl-tRNA synthetases are reported in this work. A close inspection revealed how the enzymes and the 20 gene-encoded amino acids are intertwined on the polyhedron model. Complementary and cooperative symmetries between class I and class II aminoacyl-tRNA synthetases displayed by a 28-gon organization are discussed, and we found that the two previously suggested evolutionary axes within the genetic code overlap the symmetry axes within the two classes of aminoacyl-tRNA synthetases. Moreover, it has been shown that the side-chain carbon-atom numbers (2, 1, 3, 4 and 7) in the overwhelming majority of the amino acids recognized by each of the two classes of aminoacyl-tRNA synthetases are determined by a mathematical relationship, the Lucas series. A stepwise co-evolutionary selection logic of the amino acids is manifested by the amino acid side-chain carbon-atom number balance at ‘17’, when grouping the genetic code doublets in the 28-gon organization. The number ‘17’ equals the sum of the initial five numbers in the Lucas series, which are 2, 1, 3, 4 and 7.     

Expanding genetic code: Amino acids 21 and 22, selenocysteine and pyrrolysine

The discovery of two atypical amino acids, selenocysteine and pyrrolysine, in the genetic code is discussed. These findings have expanded our understanding of the genetic code, since the repertoire of amino acids in the genetic code was supplemented by two novel ones, in addition of the standard 20 amino acids. Current views on specific mechanisms of selenocysteine insertion in forming selenoproteins are considered, as well as the results of studies of new translational components involved in biosynthesis and incorporation of selenocysteine at different stages of translation. Similarity in the strategies of decoding UGA and UAG as codons for respectively selenocysteine and pyrrolysine is discussed. The review also presents evidence on the medical and biological role of selenium and selenoproteins containing selenocysteine as the main biological form of selenium.     

Structuring of the genetic code took place at acidic pH

I have observed that in multiple regression the number of codons specifying amino acids in the genetic code is positively correlated with the isoelectric point of amino acids and their molecular weight. Therefore basic amino acids are, on average, codified in the genetic code by a larger number of codons, which seems to imply that the genetic code originated in an acidic 'intracellular' environment. Moreover, I compare the proteins from Picrophilus torridus and Thermoplasma volcanium, which have different intracellular pH and I define the ranks of acidophily for the amino acids. A simple index of acidophily (AI), which can be easily obtained from acidophily ranks, can be associated to any protein and, therefore, can also be associated to the genetic code if the number of synonymous codons attributed to the amino acids in the code is assumed to be the frequency with which the amino acids appeared in ancestral proteins. Finally, the sampling of the variable AI among organisms having an intracellular pH less than or equal to 6.6 and those having a non-acidic intracellular pH leads to the conclusion that the value of the genetic code's AI is not typical of proteins of the latter organisms. As the genetic code's AI value is also statistically not different from that of proteins of the organisms having an acidic intracellular pH, this supports the hypothesis that the structuring of the genetic code took place in acidic pH conditions.     

The Historical Factor: The Biosynthetic Relationships Between Amino Acids and Their Physicochemical Properties in the Origin of the Genetic Code

Two forces are in general, hypothesized to have influenced the origin of the organization of the genetic code: the physicochemical properties of amino acids and their biosynthetic relationships. In view of this, we have considered a model incorporating these two forces. In particular, we have studied the optimization level of the physicochemical properties of amino acids in the set of amino acid permutation codes that respects the biosynthetic relationships between amino acids. Where the properties of amino acids are represented by polarity and molecular volume we obtain indetermination percentages in the organization of the genetic code of approximately 40%. This indicates that the contingent factor played a significant role in structuring the genetic code. Furthermore, this result is in agreement with the genetic code coevolution hypothesis, which attributes a merely ancillary role to the properties of amino acids while it suggests that it was their biosynthetic relationships that organized the code. Furthermore, this result does not favor the stereochemical models proposed to explain the origin of the genetic code. On the other hand, where the properties of amino acids are represented by polarity alone, we obtain an indetermination percentage of at least 21.5%. This might suggest that the polarity distances played an important role and would therefore provide evidence in favor of the physicochemical hypothesis of genetic code origin. Although, overall, the analysis might have given stronger support to the latter hypothesis, this did not actually occur. The results are therefore discussed in the context of the different theories proposed to explain the origin of the genetic code. Received: 10 September 1996 / Accepted: 3 March 1997     

The triplet genetic code had a doublet predecessor

Information theoretic analysis of genetic languages indicates that the naturally occurring 20 amino acids and the triplet genetic code arose by duplication of 10 amino acids of class-II and a doublet genetic code having codons NNY and anticodons GNN. Evidence for this scenario is presented based on the properties of aminoacyl-tRNA synthetases, amino acids and nucleotide bases.     

Genetic code preferentially conserves long-range interactions among the amino acids

The physical properties of amino acids were investigated in order to evaluate their possible relationship to the assignment of codons for amino acids in the genetic code. A comparison of the interconversion probability between amino acids and the distances between the amino acids for individual physical properties revealed a striking hierarchy among the physical properties. Surprisingly, it is the long-range/solvent interactions and not the short-range/stereochemical properties which are preferentially conserved in the genetic code.     

The evolution of aminoacyl-tRNA synthetases,the biosynthetic pathways of amino acids and the genetic code

In this paper the partition metric is used to compare binary trees deriving from (i) the study of the evolutionary relationships between aminoacyl-tRNA synthetases, (ii) the physicochemical properties of amino acids and (iii) the biosynthetic relationships between amino acids. If the tree defining the evolutionary relationships between aminoacyl-tRNA synthetases is assumed to be a manifestation of the mechanism that originated the organization of the genetic code, then the results appear to indicate the following: the hypothesis that regards the genetic code as a map of the biosynthetic relationships between amino acids seems to explain the organization of the genetic code, at least as plausibly as the hypotheses that consider the physicochemical properties of amino acids as the main adaptive theme that lead to the structuring of the code.     

The complementarity of the chemical parameters of amino acids and anticodons in the genetic code

Chemical language of the genetic code is suggested in which elementary information code units are presented by functional groups of amino acids and nucleotides. Using this language, the existence of correspondence and conformity of chemical parameters of amino acids and of central nucleotides of their anticodons was demonstrated. These findings confirm the idea that the genetic code is determined by chemical properties of amino acids and nucleotides and that this determination is the result of direct specific interactions between amino acids and nucleotide triplets at the stage of the origin of the code. The data obtained reveal primary role of anticodon triplets in the origin of the code. Key role of the central nucleotide in triplets for amino acid coding is confirmed.     

Polarity and hydropathic properties of natural amino acids

A new classification of amino acids according to their polarity and symmetric location in the spatial structure of the genetic code is suggested. The polar amino acids are: R, S (codons AGC and AGU), K, N, Q, H, W, C, Y, G, E, D; apolar ones are: T, M, I, P, L, S (codons UCN). Polar and apolar amino acids are grouped into three families whose members possess complementarity with respect to the symmetric structure of the genetic code. Interaction of these complementary polar and apolar amino acids encodes formation of the space structures and ligand-receptor complexes of proteins. Correlation between the polar and hydropathic properties of amino acids is investigated. Normalization of 38 hydrophobicity scales of natural amino acids is carried out. A discrepancy between structures of polar/hydrophilic and apolar/hydrophobic groups of amino acids is demonstrated. According to the signature principle this discrepancy is due to different properties of amino acid side radicals which, in turn, depend on the second component of the reaction and on environmental conditions.     

Natural history and experimental evolution of the genetic code

The standard genetic code is a set of rules that relates the 20 canonical amino acids in proteins to groups of three bases in the mRNA. It evolved from a more primitive form and the attempts to reconstruct its natural history are based on its present-day features. Genetic code engineering as a new research field was developed independently in a few laboratories during the last 15 years. The main intention is to re-program protein synthesis by expanding the coding capacities of the genetic code via re-assignment of specific codons to un-natural amino acids. This article focuses on the question as to which extent hypothetical scenarios that led to codon re-assignments during the evolution of the genetic code are relevant for its further evolution in the laboratory. Current attempts to engineer the genetic code are reviewed with reference to theoretical works on its natural history. Integration of the theoretical considerations into experimental concepts will bring us closer to designer cells with target-engineered genetic codes that should open not only tremendous possibilities for the biotechnology of the twenty-first century but will also provide a basis for the design of novel life forms.     

Selection, history and chemistry: the three faces of the genetic code.

The genetic code might be a historical accident that was fixed in the last common ancestor of modern organisms. 'Adaptive', 'historical' and 'chemical' arguments, however, challenge such a 'frozen accident' model. These arguments propose that the current code is somehow optimal, reflects the expansion of a more primitive code to include more amino acids, or is a consequence of direct chemical interactions between RNA and amino acids, respectively. Such models are not mutually exclusive, however. They can be reconciled by an evolutionary model whereby stereochemical interactions shaped the initial code, which subsequently expanded through biosynthetic modification of encoded amino acids and, finally, was optimized through codon reassignment. Alternatively, all three forces might have acted in concert to assign the 20 'natural' amino acids to their present positions in the genetic code.