The case for the anticode

The present paper will focus on the developments in our lab related to the origin of the code since the Israel meeting (1,2). Principally these items are: (a) a new set of correlations (3) which include ranked hydrophobicities of amino acids and dinucleotides; (b) binding constants (4) of Phe for the four mononucleotides; and (c) binding constants (5) of Phe, Leu, Ile, Val, and Gly for polyadenylic acid (poly A). The data continue to support a model for the origin of the code based on relationships between amino acids and their anticodons.     

A new hypothesis on the evolution of the genetic code

Summary Starting from the assumption that specific steric and energetic interactions between amino acids and their respective anticodons could exist, the evolution of the genetic code is deduced from purely chemical and physical reasons. In this model the amino acids are intercalated between the two first anticodon bases and their carbon bound hydrogen atoms are assumed to penetrate into the electron clouds of the bases. By these means a gain in energy and a fixation of the amino acid is obtained in such a way that the anticodon nucleotides could be determinant for the nature of the amino acids.     

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.     

Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA(2)(Ala). This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.     

Genetic code correlations: Amino acids and their anticodon nucleotides

Summary The data here show direct correlations between both the hydrophobicity and the hydrophilicity of the homocodonic amino acids and their anticodon nucleotides. While the differences between properties of uracil and cytosine derivatives are small, further data show that uracil has an affinity for charged species. Although these data suggest that molecular relationships between amino acids and anticodons were responsible for the origin of the code, it is not clear what the mechanism of the origin might have been.     

Codon recognition patterns as deduced from sequences of the complete set of transfer RNA species in Mycoplasma capricolum. Resemblance to mitochondria

The nucleotide sequences of the complete set of tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been determined. This bacterium represents the first genetic system in which the sequences of all the tRNA species have been determined at the RNA level. There are 29 tRNA species: three for Leu, two each for Arg, Ile, Lys, Met, Ser, Thr and Trp, and one each for the other 12 amino acids as judged from aminoacylation and the anticodon nucleotide sequences. The number of tRNA species is the smallest among all known genetic systems except for mitochondria. The tRNA anticodon sequences have revealed several features characteristic of M. capricolum. (1) There is only one tRNA species each for Ala, Gly, Leu, Pro, Ser and Val family boxes (4-codon boxes), and these tRNAs all have an unmodified U residue at the first position of the anticodon. (2) There are two tRNAThr species having anticodons UGU and AGU; the first positions of these anticodons are unmodified. (3) There is only one tRNA with anticodon ICG in the Arg family box (CGN); this tRNA can translate codons CGU, CGC and CGA. No tRNA capable of translating codon CGG has been detected, suggesting that CGG is an unassigned codon in this bacterium. (4) A tRNATrp with anticodon UCA is present, and reads codon UGA as Trp. On the basis of these and other observations, novel codon recognition patterns in M. capricolum are proposed. A comparatively small total, 13, of modified nucleosides is contained in all M. capricolum tRNAs. The 5' end nucleoside of the T psi C-loop (position 54) of all tRNAs is uridine, not modified to ribothymidine. The anticodon composition, and hence codon recognition patterns, of M. capricolum tRNAs resemble those of mitochondrial tRNAs.     

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.     

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.     

Decoding of tandem quadruplets by adjacent tRNAs with eight-base anticodon loops

To expand the genetic code for specification of multiple non-natural amino acids, unique codons for these novel amino acids are needed. As part of a study of the potential of quadruplets as codons, the decoding of tandem UAGA quadruplets by an engineered tRNA^Leu with an eight-base anticodon loop, has been investigated. When GCC is the codon immediately 5′ of the first UAGA quadruplet, and release factor 1 is partially inactivated, the tandem UAGAs specify two leucines with an overall efficiency of at least 10%. The presence of a purine at anticodon loop position 32 of the tRNA decoding the codon 5′ to the first UAGA seems to influence translation of the following codon. Another finding is intraribosomal dissociation of anticodons from codons and their re-pairing to mRNA at overlapping or nearby codons. In one case where GCC is replaced by CGG, only a single Watson–Crick base pair can form upon re-pairing when decoding is resumed. This has implications for the mechanism of some cases of programmed frameshifting.     

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.     

RNA–Amino Acid Binding: A Stereochemical Era for the Genetic Code

By combining crystallographic and NMR structural data for RNA-bound amino acids within riboswitches, aptamers, and RNPs, chemical principles governing specific RNA interaction with amino acids can be deduced. Such principles, which we summarize in a “polar profile”, are useful in explaining newly selected specific RNA binding sites for free amino acids bearing varied side chains charged, neutral polar, aliphatic, and aromatic. Such amino acid sites can be queried for parallels to the genetic code. Using recent sequences for 337 independent binding sites directed to 8 amino acids and containing 18,551 nucleotides in all, we show a highly robust connection between amino acids and cognate coding triplets within their RNA binding sites. The apparent probability (P) that cognate triplets around these sites are unrelated to binding sites is ≅5.3 × 10⁻⁴⁵ for codons overall, and P ≅ 2.1 × 10⁻⁴⁶ for cognate anticodons. Therefore, some triplets are unequivocally localized near their present amino acids. Accordingly, there was likely a stereochemical era during evolution of the genetic code, relying on chemical interactions between amino acids and the tertiary structures of RNA binding sites. Use of cognate coding triplets in RNA binding sites is nevertheless sparse, with only 21% of possible triplets appearing. Reasoning from such broad recurrent trends in our results, a majority (approximately 75%) of modern amino acids entered the code in this stereochemical era; nevertheless, a minority (approximately 21%) of modern codons and anticodons were assigned via RNA binding sites. A Direct RNA Template scheme embodying a credible early history for coded peptide synthesis is readily constructed based on these observations.     

Red queen dynamics of protein translation

We explore adaptive theories for the diversity of translational binding based on the genetic code viewed as a primitive mechanism of resistance. Modifying the set of codons bound by tRNA anticodon molecules or changing the specificity of binding, reduces the replication rate of translational parasites such as viruses. Increased translational efficiency of the parasite requires a high degree of specificity of host tRNAs for the parasite codons. This suggests that the genetic code might serve as the first line of defense against infection. We construct a red queen theory for translational diversity: a theory in which host-translational strategies- as defined by the degree of redundancy (a single anticodon binding many codons for a single amino acid) or degeneracy (many anticodons binding many codons for a single amino acid)-are constantly shifting through time to evade parasitism but where neither parasite nor host gain a systematic advantage.     

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.     

Conformational relationships between amino acids and their anticodons in the primitive decoding system

Detailed calculations of the conformational characteristics of a primitive decoding system are presented. A penta-nucleotide serves as the primitive tRNA (PIT) with a triplet of primitive anticodon (PAC) in a helical conformation. This molecular moiety has a cleft in the middle. An amino acid can comfortably nestle into the cleft. The conformation of this molecular association is stabilised by a few hydrogen bonds. The stereochemistry of the moiety restricts the conformational possibilities and the sidechain of the amino acid gets oriented at a proper position and in the correct direction to interact intimately with the PAC in the middle of the PIT. The model favours L-amino acids for beta-D-ribonucleotides. The location of the sidechain of the amino acid in the PIT gives a raison d'être for the important features of the organisation of nucleotide triplets for amino acids in the Genetic Code. The interaction of a few key amino acids with the different combinations of bases as PAC sequences has been studied and the stereochemical basis for the selection of the anticodons for amino acids is elucidated.     

Studying the evolutionary relationships and phylogenetic trees of 21 groups of tRNA sequences based on complex networks

To find out the evolutionary relationships among different tRNA sequences of 21 amino acids, 22 networks are constructed. One is constructed from whole tRNAs, and the other 21 networks are constructed from the tRNAs which carry the same amino acids. A new method is proposed such that the alignment scores of any two amino acids groups are determined by the average degree and the average clustering coefficient of their networks. The anticodon feature of isolated tRNA and the phylogenetic trees of 21 group networks are discussed. We find that some isolated tRNA sequences in 21 networks still connect with other tRNAs outside their group, which reflects the fact that those tRNAs might evolve by intercrossing among these 21 groups. We also find that most anticodons among the same cluster are only one base different in the same sites when S ≥ 70, and they stay in the same rank in the ladder of evolutionary relationships. Those observations seem to agree on that some tRNAs might mutate from the same ancestor sequences based on point mutation mechanisms.     

Genetics and the origin of the genetic code

The genetic code has been analysed by a method similar to that used by Gregor Mendel. The current codon catalogue is shown to be symmetrically subdivisible into two discrete subcatalogues of eight quartets each by classifying the quartets asmonocoding (for one amino acid only) vsheterocoding (for two amino acids or for amino acid plus nonsense). The internal symmetries of the two subcatalogues are identical, and are governed by two common parity rules. These rules, together with one governing the subdivision itself, can be explained by the hypothesis that two primaeval sets of polynucleotide-borne anticodons, corresponding closely but not exactly with the subcatalogues originated independently and separately (were not originally together within any replicating pre-or proto-biont). The discorrespondence between the primaeval sets and the subcatalogues is itself symmetrical, involving quartets sharing identical locations in the two subcatalogues. The primaeval sets correspond exactly with the subdivisions of the catalogue proposed by Skoog and co-workers on the basis of the presence vs the absence of cytokinins or “cytokininlike bases” adjacent to the anticodons. A molecular model for the origin of the primaeval anticodon sets is described, and the relationship of the hypothesis with the origin of life, together with some possibilities for testing it, are discussed.     

Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Binding of misacylated tRNAs to the ribosomal A site

To test whether the ribosome displays specificity for the esterified amino acid and the tRNA body of an aminoacyl-tRNA (aa-tRNA), the stabilities of 4 correctly acylated and 12 misacylated tRNAs in the ribosomal A site were determined. By introducing the GAC (valine) anticodon into each tRNA, a constant anticodon.codon interaction was maintained, thus removing concern that different anticodon.codon strengths might affect the binding of the different aa-tRNAs to the A site. Surprisingly, all 16 aa-tRNAs displayed similar dissociation rate constants from the A site. These results suggest that either the ribosome is not specific for different amino acids and tRNA bodies when intact aa-tRNAs are used or the specificity for the amino acid side chain and tRNA body is masked by a conformational change upon aa-tRNA release.     

The origin of the genetic code: amino acids as cofactors in an RNA world.

The genetic code, understood as the specific assignment of amino acids to nucleotide triplets, might have preceded the existence of translation. Amino acids became utilized as cofactors by ribozymes in a metabolically complex RNA world. Specific charging ribozymes linked amino acids to corresponding RNA handles, which could basepair with different ribozymes, via an anticodon hairpin, and so deliver the cofactor to the ribozyme. Growing of the 'handle' into a presumptive tRNA was possible while function was retained and modified throughout. A stereochemical relation between some amino acids and cognate anticodons/codons is likely to have been important in the earliest assignments. Recent experimental findings, including selection for ribozymes catalyzing peptide-bond formation and those utilizing an amino acid cofactor, hold promise that scenarios of this major transition can be tested.