Accurate translation of the genetic code depends on tRNA modified nucleosides

Transfer RNA molecules translate the genetic code by recognizing cognate mRNA codons during protein synthesis. The anticodon wobble at position 34 and the nucleotide immediately 3' to the anticodon triplet at position 37 display a large diversity of modified nucleosides in the tRNAs of all organisms. We show that tRNA species translating 2-fold degenerate codons require a modified U(34) to enable recognition of their cognate codons ending in A or G but restrict reading of noncognate or near-cognate codons ending in U and C that specify a different amino acid. In particular, the nucleoside modifications 2-thiouridine at position 34 (s(2)U(34)), 5-methylaminomethyluridine at position 34 (mnm(5)U(34)), and 6-threonylcarbamoyladenosine at position 37 (t(6)A(37)) were essential for Watson-Crick (AAA) and wobble (AAG) cognate codon recognition by tRNA(UUU)(Lys) at the ribosomal aminoacyl and peptidyl sites but did not enable the recognition of the asparagine codons (AAU and AAC). We conclude that modified nucleosides evolved to modulate an anticodon domain structure necessary for many tRNA species to accurately translate the genetic code.     

On the origin of the genetic code and the stability of the translation apparatus

An “error theory” is developed which can be applied to determine the stability of a macromolecular translation machinery which reproduces itself. It is shown that the overall effects of a multitude of possible error versions of macromolecules can be treated statistically, and that such a statistical approach is of considerable usefulness in the theoretical treatment of complex macromolecular systems. The theory is developed within the context of a detailed treatment of the “frozen accident” hypothesis for the origin of the genetic code. A model is described which permits some thermodynamic characterization of the components involved in the code nucleation. The model also proves useful in resolving a stability “paradox” described by Orgel, which relates to the translation stability in present-day organisms and mechanisms of ageing. It indicates that any experimentally found decrease in translation accuracy with age is probably not due to an inherent instability in the translation apparatus. Relevant experiments are suggested.     

On the origin of the genetic code

It is argued that three chemical criteria determined the evolution of the genetic code: codon-anticodon pairing; codon-amino acid pairing; amino acid pairing. The first criterium determined the set of interactive nucleotides; the second, the set of nucleotides interactive with amino acids; the third, the set of mutually interactive amino acids. The code resulted from the intersection of these sets. This hypothesis explains the specificity and universality of the code as well as the “choice” of the standard amino acids and nucleotides from among those available in nature. The specific mechanism for codon-amino acid pairing assumed here is the “backwards” (Crick, 1967) Pelc-Welton (1966) models. Three types of evidence support “backwards” pairing: parallel genetic coding of amino acid pairs (Root-Bernstein, 1982); results of binding experiments by Saxinger and Ponnamperuma (1974); reinterpretation of Jungck's (1978) correlations between the properties of amino acids and their respective anticodon nucleotides. The inversion of the code to its present state occurred as a result of the evolution of tRNA molecules which supplanted parallel codon-amino acid interactions with antiparallel codon-anticodon ones. The paper concludes with suggestions for testing the hypothesis.     

tRNA genes and the genetic code

The genetic code describes translational assignments between codons and amino acids. tRNAs and aminoacyl-tRNA synthetases (aaRSs) are those molecules by means of which these assignments are established. Any aaRS recognizes its tRNAs according to some of their nucleotides called identity elements (IEs). Let a 1Mut-similarity Sim (1Mut) be the average similarity between such tRNA genes whose codons differ by one point mutation. We showed that: (1) a global maximum of Sim (1Mut) is reached at the standard genetic code 27 times for 4 sets of IEs of tRNA genes of eukaryotic species, while it is so only 5 times for similarities Sim (C&R) between all tRNA genes whose codons lie in the same column or row of the code. Therefore, point mutations of anticodons were tested by nature to recruit tRNAs from one isoaccepting group to another, (2) because plain similarities Sim (all) between tRNA genes of species within any of the three domains of life are higher than between tRNA genes of species belonging to different domains, tRNA genes retained information about early evolution of cells, (3) we searched the order of tRNAs in which they were most probably assigned to their codons and amino acids. The beginning Ala, (Val), Pro, Ile, Lys, Arg, Trp, Met, Asp, Cys, (Ser) of our resulting chronology lies under a plateau on a graph of Sim (1Mut,IE)(univ.ancestors) plotted over this chronology for a set S(IE) of all IEs of tRNA genes, whose universal ancestors were separately computed for each codon. This plateau has remained preserved along the whole line of evolution of the code and is consistent with observations of Ribas de Pouplana and Schimmel [2001. Aminoacy1-tRNA synthetases: potential markers of genetic code development. Trends Biochem. Sci. 26, 591-598] that specific pairs of aaRSs-one from each of their two classes-can be docked simultaneously onto the acceptor stem of tRNA and hence an interaction existed between their ancestors using a reduced code, (4) sharpness of a local maximum of Sim (1Mut) at the standard code is almost 100% along our chronologies.     

On the origin and evolution of the genetic code

Two ideas have essentially been used to explain the origin of the genetic code: Crick's frozen accident and Woese's amino acid-codon specific chemical interaction. Whatever the origin and codon-amino acid correlation, it is difficult to imagine the sudden appearance of the genetic code in its present form of 64 codons coding for 20 amino acids without appealing to some evolutionary process. On the contrary, it is more reasonable to assume that it evolved from a much simpler initial state in which a few triplets were coding for each of a small number of amino acids. Analysis of genetic code through information theory and the metabolism of pyrimidine biosynthesis provide evidence that suggests that the genetic code could have begun in an RNA world with the two letters A and U grouped in eight triplets coding for seven amino acids and one stop signal. This code could have progressively evolved by making gradual use of letters G and C to end with 64 triplets coding for 20 amino acids and three stop signals. According to proposed evidence, DNA could have appeared after the four-letter structure was already achieved. In the newborn DNA world, T substituted U to get higher physicochemical and genetic stability.     

On the possible origin and evolution of the genetic code

The genetic code is examined for indications of possible preceding codes that existed during early evolution. Eight of the 20 amino acids are coded by ‘quartets’ of codons with four-fold degeneracy, and 16 such quartets can exist, so that an earlier code could have provided for 15 or 16 amino acids, rather than 20. If two-fold degeneracy is postulated for the first position of the codon, there could have been 10 amino acids in the code. It is speculated that these may have been phenylalanine, valine, proline, alanine, histidine, glutamine, glutamic acid, aspartic acid, cysteine and glycine. There is a notable deficiency of arginine in proteins, despite the fact that it has six codons. Simultaneously, there is more lysine in proteins than would be expected from its two codons, if the four bases in mRNA are equiprobable and are arranged randomly. It is speculated that arginine is an ‘intruder’ into the genetic code, and that it may have displaced another amino acid such as ornithine, or may even have displaced lysine from some of its previous codon assignments. As a result, natural selection has favored lysine against the fact that it has only two codons. The introduction of tRNA into protein synthesis may have been a cataclysmic and comparatively sudden event, since duplication of tRNA takes place readily, and point mutations could rapidly differentiate members of the family of duplicates from each. Two tRNAs for different amino acids may have a common ancestor that existed more recently than the separation of the prokaryotes and eukaryotes. This is shown by homology of twoE. coli tRNAs for glycine and valine, and two yeast tRNAs for arginine and lysine.     

The origin of the genetic code

A new approach to the origin of the genetic code is proposed based on some regularities in the nucleotide distribution pattern of the code. The relative amounts of various amino acids in primitive proteins were possibly different from those in organisms living today. The primordial ratio was supposed to shift to the modern one guided by the action of primitive nucleotides. Each primitive tRNA had a discriminator site and, distinguished from it, an anticodon site. It is also postulated that primordially each amino acid could correspond to a wide variety of codons. During the course of the evolutionary change, a selective mechanism worked among the protobionts so that less frequent nucleotides became associated with more abundant amino acids in the primordial conditions, thus finally leading to the present codon catalogue.Presented at The International Seminar: The Origin of Life held in Moscow, August 2–7, 1974.     

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.     

The genetic code and the origin of life

The problem of the origin of life understandably counts as one of the most exciting questions in the natural sciences, but in spite of almost endless speculation on this subject, it is still far from its final solution. The complexity of the functional correlation between recent nucleic acids and proteins can e.g. give rise to the assumption that the genetic code (and life) could not originate on the Earth. It was Portelli (1975) who published the hypothesis that the genetic code could not originate during the history of the Earth. In his opinion the recent genetic code represents the informational message transmitted by living systems of the previous cycle of the Universe. Here however, we defend the existence of a certain strategy in the syntheses of the genetic code during the history of the Earth. The strategy of correlation between amino acid and nucleotide polymers made an increasing velocity of the chemical evolution possible, that is, it increased the velocity of formation of the genetic code. Thus, life with the recent genetic code could originate on the Earth within the present cycle of the Universe.Present address: Institute for Pharmacy and Biochemistry, 533 51 Pardubice, Czechoslovakia.