Phenylalanine-Binding RNAs and Genetic Code Evolution

We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets. Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70% of the 105 sequenced RNAs. The K _D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites. However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10⁻¹¹. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg, Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during later code evolution.     

The Evolution of the Genetic Code Revisited

The evolution of the genetic code in terms of the adoption of new codons has previously been related to the relative thermostability of codon–anticodon interactions such that the most stable interactions have been hypothesised to represent the most ancient coding capacity. This derivation is critically dependent on the accuracy of the experimentally determined stability parameters. A new set of parameters recently determined for B-DNA reveals that the codon–anticodon pairs for the codes in non-plant mitochondria on the one hand and prokaryotic and eukaryotic organisms on the other can be unequivocally divided into two classes – the most stable base steps define a common code specified by the first two bases in a codon while the less stable base steps correlate with divergent usage and the adoption of a 3-letter code. This pattern suggests that the fixation of codons for A, G, P, V, S, T, D/E, R may have preceded the divergence of the non-plant mitochondrial line from other organisms. Other variations in the code correlate with the least stable codon–anticodon pairs. Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005.     

On the Origin of Frameshift-Robustness of the Standard Genetic Code

The standard genetic code (SGC) has been extensively analyzed for the biological ramifications of its nonrandom structure. For instance, mismatch errors due to point mutation or mistranslation have an overall smaller effect on the amino acid polar requirement under the SGC than under random genetic codes (RGCs). A similar observation was recently made for frameshift errors, prompting the assertion that the SGC has been shaped by natural selection for frameshift-robustness—conservation of certain amino acid properties upon a frameshift mutation or translational frameshift. However, frameshift-robustness confers no benefit because frameshifts usually create premature stop codons that cause nonsense-mediated mRNA decay or production of nonfunctional truncated proteins. We here propose that the frameshift-robustness of the SGC is a byproduct of its mismatch-robustness. Of 564 amino acid properties considered, the SGC exhibits mismatch-robustness in 93–133 properties and frameshift-robustness in 55 properties, respectively, and that the latter is largely a subset of the former. For each of the 564 real and 564 randomly constructed fake properties of amino acids, there is a positive correlation between mismatch-robustness and frameshift-robustness across one million RGCs; this correlation arises because most amino acid changes resulting from a frameshift are also achievable by a mismatch error. Importantly, the SGC does not show significantly higher frameshift-robustness in any of the 55 properties than RGCs of comparable mismatch-robustness. These findings support that the frameshift-robustness of the SGC need not originate through direct selection and can instead be a site effect of its mismatch-robustness.     

Evolution of Genetic Alphabet and of Amino Acid Code

On considering chemical evolution of the Earth since the time of its appearance when its composition was similar to the elementary composition of star substance, a tentative hypothesis has been put forward that molecular evolution of the four-letter genetic alphabet includes two periods: I (pre-oxygen) and II (oxygenated) periods of chemical evolution. At the period I, in the primary Earth atmosphere the first nitrogen base, adenine (A), containing no oxygen appeared. The period II, during which three other nitrogen bases appeared in the atmosphere, consisted of three stages; at the first stage, guanine (G) appeared, at the second, cytosine (C), and at the third stage, uracyl (U). In accordance with the above periods, formation of codons and amino acids in nature was taking place presumably by the following way: at the period I, the first and the only codon AAA appeared, to which the amino acid lysine (Lys) corresponded; at the first stage of the period II, 7 codons and 3 amino acids (Arg, Glu, Gly) appeared; at the second stage, 19 codons and 8 new amino acids (Asn, Gin, Ser, Asp, Thr, Ala, His, Pro) appeared; at the third stage, 37 codons and more 8 new amino acids (Trp, Tyr, Cys, Ile, Met, Val, Leu, Phe) appeared. Thereby, in the course of biochemical evolution, 20 amino acids and 64 codons appeared in nature.     

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.     

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.     

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.     

An Extended RNA Code and its Relationship to the Standard Genetic Code: An Algebraic and Geometrical Approach

An algebraic and geometrical approach is used to describe the primaeval RNA code and a proposed Extended RNA code. The former consists of all codons of the type RNY, where R means purines, Y pyrimidines, and N any of them. The latter comprises the 16 codons of the type RNY plus codons obtained by considering the RNA code but in the second (NYR type), and the third, (YRN type) reading frames. In each of these reading frames, there are 16 triplets that altogether complete a set of 48 triplets, which specify 17 out of the 20 amino acids, including AUG, the start codon, and the three known stop codons. The other 16 codons, do not pertain to the Extended RNA code and, constitute the union of the triplets YYY and RRR that we define as the RNA-less code. The codons in each of the three subsets of the Extended RNA code are represented by a four-dimensional hypercube and the set of codons of the RNA-less code is portrayed as a four-dimensional hyperprism. Remarkably, the union of these four symmetrical pairwise disjoint sets comprises precisely the already known six-dimensional hypercube of the Standard Genetic Code (SGC) of 64 triplets. These results suggest a plausible evolutionary path from which the primaeval RNA code could have originated the SGC, via the Extended RNA code plus the RNA-less code. We argue that the life forms that probably obeyed the Extended RNA code were intermediate between the ribo-organisms of the RNA World and the last common ancestor (LCA) of the Prokaryotes, Archaea, and Eucarya, that is, the cenancestor. A general encoding function, E, which maps each codon to its corresponding amino acid or the stop signal is also derived. In 45 out of the 64 cases, this function takes the form of a linear transformation F, which projects the whole six-dimensional hypercube onto a four-dimensional hyperface conformed by all triplets that end in cytosine. In the remaining 19 cases the function E adopts the form of an affine transformation, i.e., the composition of F with a particular translation. Graphical representations of the four local encoding functions and E, are illustrated and discussed. For every amino acid and for the stop signal, a single triplet, among those that specify it, is selected as a canonical representative. From this mapping a graphical representation of the 20 amino acids and the stop signal is also derived. We conclude that the general encoding function E represents the SGC itself.     

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...     

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     

On the Evolution of the Standard Genetic Code: Vestiges of Critical Scale Invariance from the RNA World in Current Prokaryote Genomes

Herein two genetic codes from which the primeval RNA code could have originated the standard genetic code (SGC) are derived. One of them, called extended RNA code type I, consists of all codons of the type RNY (purine-any base-pyrimidine) plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. In order to test if putative nucleotide sequences in the RNA World and in both extended RNA codes, share the same scaling and statistical properties to those encountered in current prokaryotes, we used the genomes of four Eubacteria and three Archaeas. For each prokaryote, we obtained their respective genomes obeying the RNA code or the extended RNA codes types I and II. In each case, we estimated the scaling properties of triplet sequences via a renormalization group approach, and we calculated the frequency distributions of distances for each codon. Remarkably, the scaling properties of the distance series of some codons from the RNA code and most codons from both extended RNA codes turned out to be identical or very close to the scaling properties of codons of the SGC. To test for the robustness of these results, we show, via computer simulation experiments, that random mutations of current genomes, at the rates of 10⁻¹⁰ per site per year during three billions of years, were not enough for destroying the observed patterns. Therefore, we conclude that most current prokaryotes may still contain relics of the primeval RNA World and that both extended RNA codes may well represent two plausible evolutionary paths between the RNA code and the current SGC.     

Genetic Hotels for the Standard Genetic Code: Evolutionary Analysis Based upon Novel Three-Dimensional Algebraic Models

Herein, we rigorously develop novel 3-dimensional algebraic models called Genetic Hotels of the Standard Genetic Code (SGC). We start by considering the primeval RNA genetic code which consists of the 16 codons of type RNY (purine-any base-pyrimidine). Using simple algebraic operations, we show how the RNA code could have evolved toward the current SGC via two different intermediate evolutionary stages called Extended RNA code type I and II. By rotations or translations of the subset RNY, we arrive at the SGC via the former (type I) or via the latter (type II), respectively. Biologically, the Extended RNA code type I, consists of all codons of the type RNY plus codons obtained by considering the RNA code but in the second (NYR type) and third (YRN type) reading frames. The Extended RNA code type II, comprises all codons of the type RNY plus codons that arise from transversions of the RNA code in the first (YNY type) and third (RNR) nucleotide bases. Since the dimensions of remarkable subsets of the Genetic Hotels are not necessarily integer numbers, we also introduce the concept of algebraic fractal dimension. A general decoding function which maps each codon to its corresponding amino acid or the stop signals is also derived. The Phenotypic Hotel of amino acids is also illustrated. The proposed evolutionary paths are discussed in terms of the existing theories of the evolution of the SGC. The adoption of 3-dimensional models of the Genetic and Phenotypic Hotels will facilitate the understanding of the biological properties of the SGC.     

Evolution of the Genetic Triplet Code via Two Types of Doublet Codons

Explaining the apparent non-random codon distribution and the nature and number of amino acids in the ‘standard’ genetic code remains a challenge, despite the various hypotheses so far proposed. In this paper we propose a simple new hypothesis for code evolution involving a progression from singlet to doublet to triplet codons with a reading mechanism that moves three bases each step. We suggest that triplet codons gradually evolved from two types of ambiguous doublet codons, those in which the first two bases of each three-base window were read (‘prefix’ codons) and those in which the last two bases of each window were read (‘suffix’ codons). This hypothesis explains multiple features of the genetic code such as the origin of the pattern of four-fold degenerate and two-fold degenerate triplet codons, the origin of its error minimising properties, and why there are only 20 amino acids. Reviewing Editor: Dr. Laura Landweber An erratum to this article can be found at .     

Genetic Code Variations in Mitochondria: tRNA as a Major Determinant of Genetic Code Plasticity

Characteristic features of tRNA such as the anticodon sequence and modified nucleotides in the anticodon loop are thought to be crucial effectors for promoting or restricting codon reassignment. Our recent findings on basepairing rules between anticodon and codon in various metazoan mitochondria suggest that the complete loss of a codon is not necessarily essential for codon reassignment to take place. We postulate that a possible competition between two tRNAs with cognate anticodon sequences towards the relevant codon to be varied has a potential role in codon reassignment. Our proposition can be viewed as an expanded version of the codon capture theory proposed by Osawa and Jukes (J Mol Evol 28: 271–278, 1989). Received: 28 December 2000 / Accepted: 12 March 2001