Possible evolutionary steps in the genetic code

In mammalian mitochondrial codes, fourfold codons wobble-pair with UNN anticodons so that U wobbles with U, C, A and G. Twofold pyrimidine-terminated codons pair with GNN and twofold purine-terminated codons pair with UNN. These properties enable a prediction to be made for evolution of the universal genetic code. It was postulated (1) that an archetypal code of 16 quartets coded for 15 amino acids. If this code used UNN anticodons, then duplication of tRNA genes, followed by mutations in the anticodons and aminoacylation sites, would give rise to the present universal code.     

Codon–anticodon interaction and the genetic code evolution

The evolution of the genetic code, with 20 amino acids encoded from the beginning, is analyzed from the viewpoint of codon–anticodon interaction. Imposing a minimum principle for the interaction, in the framework of the so called crystal basis model of the genetic code, we determine the structure of the anticodons in the ancient, archetypal and early genetic codes, that are all reconciled in a unique frame. Most of our results agree with the generally accepted scheme.     

Genetic code from tRNA point of view

The possible codon-anticodon pairings follow the standard genetic code, yet in a different mode. The corresponding rules for decoding sequence of the codons in mRNA with tRNA may be called "tRNA code". In this paper we analyse the mutational and translational stability of such tRNA code. Our approach is based on the model of "ambiguous intermediate" and on the study of underlying block structure and Eulerean graph technique. It is shown that the wobble rules and the reduced number of tRNA anticodons strongly affect the mutational and translational stability of the code. The selection of tRNA anticodons, besides the optimization of translation, also ensures the more reliable start and, to a lesser extent, the stop of translation. The attribution of tRNA anticodons to the groups [WWW, WWS, SWW, SWS] and [SSS, SSW, WSS, WSW] as well as [MMM, MMK, KMM, KMK] and [KKK, KKM, MKK, MKM] clearly correlates with class I and class II aminoacyl-tRNA synthetases and obeys the principle of the optimal coding in both cases. Both W-S and M-K groupings also refer to the encoding of amino acids with the large and small side-chain volumes, which may provide such an attribution. The higher variability of tRNA code agrees with the suggestions that the variations in an assignment of tRNA anticodons may serve as the driving force generating the different variants of the genetic code.     

Unambiguous correspondence and complementarity of the chemical properties of amino acids and anticodons of the genetic code

The chemical language of genetic code is proposed. As a result of chemical language application for the analysis of the modern genetic code, the existence of an unambiguous correspondence between the chemical properties of amino acids and their coding triplets (codons and anticodons) is shown. This confirms the hypothesis of the code chemical determination. The complementarity between the chemical properties of amino acids and their anticodons (but not the codons) has been found also to exist. This observation supports the hypothesis of the genetic code determination by the direct recognition and also underlines the primary role of anticodon in the origin of genetic code in comparison with codons.     

Origin of the genetic code: first aminoacyl-tRNA synthetases could replace isofunctional ribozymes when only the second base of codons was established

Analysis of the updated compilation of more than 8,000 tRNA gene sequences confirmed our previously reported finding that in pairs of consensus tRNAs with complementary anticodons, their second bases in the acceptor stems are also complementary. This dual complementarity points to the following: (1) the operational code embodied in the acceptor stem, and the classic genetic code embodied in the anticodon could have had the same common ancestor; (2) new tRNAs most likely entered primitive translation in pairs with complementary anticodons; and (3) this process of code expansion was directed by the primordial double-strand coding. However, we did not find the dual complementarity when testing all tRNA pairs in which anticodons were complementary only at the central position, but not complementary at least at one of the flanking two positions. This observation, together with certain additional evidence, suggests that both codes were still being shaped (with only the second base established at the time) when the first protein aminoacyl-tRNA synthetases could have already started replacing their ribozymic precursors.     

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.     

Partitioning of aminoacyl-tRNA synthetases in two classes could have been encoded in a strand-symmetric RNA world

The "chicken-or-egg" dilemma dictates that archaic tRNAs be aminoacylated by ribozymic aminoacyl-tRNA synthetases, rAARSs, with protein synthetases (pAARSs) emerging later and, strikingly in two versions. However, the distribution of these two versions among the codons also suggests their involvement in development of the genetic code. Here we propose a solution to this controversy, which relies on a primordial complementarity hypothesis that in a strand-symmetric RNA world both complementary replicas of many genes could encode the first proteins. Accordingly, if one rearranges the code table in a manner that puts complementary codons directly against each other, an almost perfect mirror symmetry in tRNA aminoacylation by the two groups of synthetases is revealed. Specifically, the pairs of complementary anticodons from the same pAARS class tend to contain RR and YY dinucleotides at first and second versus third and second positions, whereas in pairs of pAARSs from the different classes these positions are occupied by YR and RY, including CG, GC, UA, and AU palindromes. The latter are indistinguishable in complementary anticodons, thus leading to erroneous aminoacylation (note that there is no such problem for RR- and YY-containing complementary anticodons). This can be averted by "spreading out" tRNA recognition by two rAARSs away from the anticodons in the opposite directions, giving two complementary rAARSs. The principle of evolutionary continuity suggests that their protein successors also arose on complementary strands. Our analyses support this hypothesis.     

Codon-acticodon recognition in the valine codon family.

An in vitro protein-synthesizing system completely dependent on added valine tRNA (valyl-tRNAval) and programmed with RNA from the phage MS2 has been used to investigate the incorporation into MS2 coat protein of valine from isoaccepting valyl-tRNAsval with the anticodons U AC (U represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC in response to the four valine codons GUU, GUC, GUA, and GUG. By examining the incorporation of valine into NH2-terminal and internal positions of three tryptic peptides from the MS2 coat protein it has been established that these anticodons each recognize all four valine codons. We therefore conclude that under our conditions of in vitro protein synthesis the genetic code, as far as the valine codons are concerned, is operationally a two letter code, i.e. the third codon nucleotide has no absolute discriminating function.     

Translation of both complementary strands might govern early evolution of the genetic code

The updated structural and phylogenetic analyses of tRNA pairs with complementary anticodons provide independent support for our earlier finding, namely that these tRNA pairs concertedly show complementary second bases in the acceptor stem. Two implications immediately follow: first, that a tRNA molecule gained its present, complete, cloverleaf shape via duplication(s) of a shorter precursor. Second, that common ancestry is shared by two major components of the genetic code within the tRNA molecule--the classic code per se embodied in anticodon triplets, and the operational code of aminoacylation embodied primarily in the first three base pairs of the acceptor stems. In this communication we show that it might have been a double, sense-antisense, in-frame translation of the very first protein-encoding genes that directed the code's earliest expansion, thus preserving this fundamental dual-complementary link between acceptors and anticodons. Furthermore, the dual complementarity appears to be consistent with two mirror-symmetrical modes by which class I and II aminoacyl-tRNA synthetases recognize the cognate tRNAs--from the minor and major groove side of the acceptor stem, respectively.     

Evolutionary changes in the genetic code

The genetic code has been influenced by directional mutation pressure affecting the base composition of DNA, sometimes in the direction of increased GC content and at other times, in the direction of AT. Such pressure led to changes in species-specific usages of codons and tRNA anticodons, and also in amino acid assignments of codons in mitochondria and in several intact organisms. These code changes are probably recent evolutionary events. The genetic code is not 'frozen', but instead it is still evolving.     

On the origin of the translation system and the genetic code in the RNA world by means of natural selection, exaptation, and subfunctionalization

Background  

The origin of the translation system is, arguably, the central and the hardest problem in the study of the origin of life, and one of the hardest in all evolutionary biology. The problem has a clear catch-22 aspect: high translation fidelity hardly can be achieved without a complex, highly evolved set of RNAs and proteins but an elaborate protein machinery could not evolve without an accurate translation system. The origin of the genetic code and whether it evolved on the basis of a stereochemical correspondence between amino acids and their cognate codons (or anticodons), through selectional optimization of the code vocabulary, as a "frozen accident" or via a combination of all these routes is another wide open problem despite extensive theoretical and experimental studies. Here we combine the results of comparative genomics of translation system components, data on interaction of amino acids with their cognate codons and anticodons, and data on catalytic activities of ribozymes to develop conceptual models for the origins of the translation system and the genetic code.     

On concerted origin of transfer RNAs with complementary anticodons

Pairs of antiparallelly oriented consensus tRNAs with complementary anticodons show surprisingly small numbers of mispairings within the 17-bp-long anticodon stem and loop region. Even smaller such complementary distances are shown by illegitimately complementary anticodons, i.e. those with allowed pairing between G and U bases. Accordingly, we suppose that transfer RNAs have emerged concertedly as complementary strands of primordial double helix-like RNA molecules. Replication of such molecules with illegitimately complementary anticodons might generate new synonymous codons for the same pair of amino acids. Logically, the idea of tRNA concerted origin dictates very ancient establishment of direct links between anticodons and the type of amino acids with which pre-tRNAs were to be charged. More specifically, anticodons (first of all, the 2nd base) could selectively target their amino acids, reaction of acylating itself being performed by another non-specific site of pre-tRNA or even by another ribozyme. In all, the above findings and speculations are consistent to the hypercyclic concept (Eigen and Schuster, 1979), and throw new light on the genetic code origin and associated problems. Also favoring this idea are data on complementary codon usage patterns in different genomes.     

Theβ-sheets of proteins,the biosynthetic relationships between amino acids,and the origin of the genetic code

Two forces are generally hypothesised as being responsible for conditioning the origin of the organization of the genetic code: the physicochemical properties of amino acids and their biosynthetic relationships (relationships between precursor and product amino acids). If we assume that the biosynthetic relationships between amino acids were fundamental in defining the genetic code, then it is reasonable to expect that the distribution of physicochemical properties among the amino acids in precursor-product relationships cannot be random but must, rather, be affected by some selective constraints imposed by the structure of primitive proteins. Analysis shows that measurements representing the size of amino acids, e.g. bulkiness, are specifically associated to the pairs of amino acids in precursor-product relationships. However, the size of amino acids cannot have been selected per se but, rather, because it reflects the-sheets of proteins which are, therefore, identified as the main adaptive theme promoting the origin of genetic code organization. Whereas there are no traces of the-helix in the genetic code table.The above considerations make it necessary to re-examine the relationship linking the hydrophilicity of the dinucleoside monophosphates of anticodons and the polarity and bulkiness of amino acids. It can be concluded that this relationship seems to be meaningful only between the hydrophilicity of anticodons and the polarity of amino acids. The latter relationship is supposed to have been operative on hairpin structures, ancestors of the tRNA molecule. Moreover, it is on these very structures that the biosynthetic links between precursor and product amino acids might have been achieved, and the interaction between the hydrophilicity of anticodons and the polarity of amino acids might have had a role in the concession of codons (anticodons) from precursors to products.     

RNA minihelices and the decoding of genetic information

The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetase. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity.     

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.     

A non-canonical genetic code in an early diverging eukaryotic lineage.

The nearly invariant nature of the ''Universal Genetic Code'' attests to its early establishment in evolution and to the difficulty of altering it now, since so many molecules are required for, and depend upon, faithful translation. Nevertheless, variations on the universal code are known in a handful of genomes. We have found one such variant in diplomonads, an early-diverging eukaryotic lineage. Genes for alpha-tubulin, beta-tubulin and elongation factor 1 alpha (EF-1alpha) from two unclassified strains of Hexamitidae were found to contain TAA and TAG (TAR) triplets at positions suggesting a variant code in which TAR codes for glutamine. We found confirmation of this hypothesis by identifying genes encoding glutamine-tRNAs with CUA and UUA anticodons. The alpha-tubulin and EF-1alpha genes from two other diplomonads, Spironucleus muris and Hexamita inflata, were also sequenced and shown to contain no such non-canonical codons. However, tRNA genes with the anticodons UUA and CUA were found in H.inflata, suggesting that this diplomonad also uses these codons, albeit infrequently. The high GC content of these genomes and the presence of two isoaccepting tRNAs compound the difficulty of understanding how this variant code arose by strictly neutral means.     

Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity.     

One ancestor for two codes viewed from the perspective of two complementary modes of tRNA aminoacylation

Background  

The genetic code is brought into action by 20 aminoacyl-tRNA synthetases. These enzymes are evenly divided into two classes (I and II) that recognize tRNAs from the minor and major groove sides of the acceptor stem, respectively. We have reported recently that: (1) ribozymic precursors of the synthetases seem to have used the same two sterically mirror modes of tRNA recognition, (2) having these two modes might have helped in preventing erroneous aminoacylation of ancestral tRNAs with complementary anticodons, yet (3) the risk of confusion for the presumably earliest pairs of complementarily encoded amino acids had little to do with anticodons. Accordingly, in this communication we focus on the acceptor stem.