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Reflections on the Origin of the Genetic Code: a Hypothesis
J. theor. Biol. (1998) 191, 191–196
Reflections on the Origin of the Genetic Code: a Hypothesis M D G International Institute of Genetics and Biophysics, CNR, Via G. Marconi 10, 80125 Naples, Italy (Received on 8 September 1997, Accepted on 29 October 1997)
The origin of the organisation of the genetic code reflects both the biosynthetic relationships between amino acids and the physicochemical interactions between these and anticodons; moreover, these two forces do not act independently. It must therefore be explained why it is simultaneously true that the anticodons of product amino acids were assigned prior to their biosynthetic appearance and that physicochemical correlations must exist between anticodons and amino acids. This gives rise to difficulties of interpretation, even of a more general nature, in the theories that have been proposed to explain the origin of the genetic code; a hypopthesis is thus presented in this manuscript. In particular, the hypothesis suggests that RNA hairpin structures, the ancestors of tRNAs, housing anticodon-like nucleotides in the stem were charged with precursor amino acids. As the precursor amino acids gradually developed into product amino acids, a coevolution came into being between the development of amino acids and that of anticodons, with a concomitant formation of the complete tRNA molecule through direct duplication of the hairpin structures. All this led to the definition of the genetic code organisation. Furthermore, this made it possible for the evolving anticodons to select the emerging product amino acids, which the hypothesis therefore considers to be unspecified in the initial phase of genetic code evolution but which were selected also because of their ability to interact with anticodons. In this way the main obstacle to interpretation is removed. Fossils of these events can now be observed in some amino acid-modified nucleosides specifically located in the tRNA anticodon loops. This is all presented in the framework of a general discussion of the ideas and data in favour of a late origin of the genetic code, as opposed to an early origin in the context of the theories proposed to explain the origin of the genetic code organisation. 7 1998 Academic Press Limited
Introduction A reflection on the time events that led to the origin of the genetic code organisation could provide information useful both for its origin and, more generally, for the origin of life. On the whole, the origin of genetic code organisation might have taken place in an early or a late stage of the origin of life. An early origin of the genetic code organisation has several implications. In view of the considerable complexity of the translation apparatus, the early origin of the genetic code implies that the code was in a very different form from its current one. In other words, the genetic code must have been pre-adapted 0022–5193/98/060191 + 06 $25.00/0/jt970580
to perform other functions which culminated in the genetic code structure proper only with their further evolution. It also seems clear that an early origin might have required some essential aspects of the genetic code to be already in play, such as anticodons or codons per se and amino acids (or some of them). In turn, this seems to imply that a stereochemical interaction between anticodons or codons and amino acids was operative at this stage of genetic code origin. Therefore, an early origin of the genetic code seems to be more consonant with the stereochemical theory of genetic code origin (Woese, 1967) or with part of the physicochemical one (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; 7 1998 Academic Press Limited
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Lacey et al., 1992). More explicitly, if the origin of the genetic code was early this can probably be attributed to stereochemical and/or physicochemical interactions between anticodons or codons and amino acids because these interactions should have been more naturally manifested in such an early stage. Alternatively, a late origin of the genetic code assumes that the code evolved in a system that was already somewhat complex. It is likely that, under the hypothesis of late genetic code origin, the stereochemical hypothesis and part of the physicochemical one lose part of their importance because the system had somehow already abandoned strict physicochemical determinism. On the other hand, the coevolution theory of genetic code origin (Wong, 1975), which is based on the strict parallelism of the biosynthetic relationships between amino acids and the organisation of the genetic code (for references, see Di Giulio, 1997), is more consonant with a late origin of the genetic code as this theory suggests that the organisation of the genetic code was established with the appearance of the biosynthetic pathways of amino acids, which does not intuitively seem to have been a very early event in the origin of life. It was therefore decided to analyse the time events that occurred during the origin of the genetic code, with the aid of the following weak proposition: Since the co-operation of numerous molecules is needed for the achievement of the genetic code, even if it was pre-adapted to perform some other function, it cannot be expected to have an early origin because even the essential elements of the translation apparatus, i.e. aminoacylated tRNAs and mRNAs, imply that the code must have undergone a long evolution which, in turn could mean that the structuring of the genetic code is probably a relatively recent invention of evolution. Therefore, in the present paper ideas and data in favour of the early and the late origin of the genetic code are discussed and the implications and consequences that this has on the theories that have been proposed to explain the origin of genetic code organisation. An Early Origin of the Genetic Code: was There an Ancient Code Based on Anticodon–Amino Acid Interactions? There is a considerable quantity of literature that points to the existence of a relationship between the physicochemical properties of amino acids and the organisation of the genetic code (for references, see Szathmary, 1993; Di Giulio, 1997). The origin of the genetic code might therefore be the direct conse-
quence of an interaction between anticodons or codons and amino acids. In actual fact, in this huge volume of literature there are a few papers that report a correlation between the properties of anticodons and those of amino acids (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; Lacey et al., 1992; Di Giulio, 1996). Other evidence is given by numerous stereochemical models based on interactions between anticodons or codons (or complexes containing them) and amino acids (Pelc & Welton, 1966; Melcher, 1974; Balasubramanian et al., 1980; Hendry et al., 1981; Shimizu, 1982; Yarus, 1991). Moreover, a stereoselective interaction between arginine and codons that codify it in an RNA has been described (Yarus, 1991) and this is therefore, in general, in favour of these models. However, one feels that it is above all the studies conducted on the tRNA identity determinants which seem to provide considerable evidence in favour of the existence of an ancient code positioned in the stem of the hairpin structures (Hopfield, 1978; de Duve, 1988; Moller & Janssen 1992; Mursier-Forsyth & Schimmel, 1993; Schimmel & Henderson, 1994; Di Giulio, 1995; Schimmel, 1995), considered to be the ancestors of the tRNAs (Hopfield, 1978; Eigen & Winkler-Oswatitsch, 1981; Bloch et al., 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994), which imply that the origin of the genetic code might have been based on interactions between anticodons (or codons) and amino acids. In this connection, the observations of Shimizu (1995) also seem to be important as the author finds that hairpin structures housing anticodons in the stem are, in the presence of a dipeptide, specifically charged with the corresponding amino acid. All these and other observations (for references, see Szathmary, 1993; Di Giulio, 1997) suggest that anticodon–amino acid interactions contributed to defining the organisation of the genetic code. It also seems natural to postulate that these interactions were expressed early in the evolution of the code as they are based on what seems to be a strict physicochemical determinism. This leads to the conclusion that there must have been an early phase in which there was a physicochemical correspondence between anticodons (or codons) and amino acids and that, therefore, there might have been a more ancient code based on stereochemical and/or physicochemical interactions. One feels that this conclusion has a very limited validity because it seems to be susceptible to the following two criticisms. (i) In my opinion the origin of the genetic code must first of all reflect aspects of the protein structure because the code evolved in order to build the latter. There are some observations
: suggesting that this might well have been the case (Jurka & Smith, 1987a, b; Di Giulio, 1996). It is also clear that the overlapping of a genetic code reflecting the structural motifs of proteins on the more ancient code based on anticodon–amino acid interactions might have removed many aspects of the latter because the languages of these two codes do not seem to be related. (ii) If there was an ancient genetic code based on anticodon–amino acid interactions before the biosynthetic pathways of amino acids also affected its organisation, what was the result of the successive action of these two forces? Here too it seems that the overlapping of a genetic code reflecting the biosynthetic relationships between amino acids on the one based on anticodon–amino acid interactions might have removed much of the ancient code. Indeed, the biosynthetic relationships between amino acids do not seem to be in the least affected by a pre-existing anticodon–amino acid interaction and these relationships might have even led to the introduction of new amino acids not codified in these interactions. The conclusion reached is that there is some doubt that a more ancient code based on anticodon–amino acid interactions even existed and therefore, in order to take these interactions into account as well, we have to hypothesise that these interactions took place at the same time as the biosynthetic transformations between amino acids. A Late Origin of the Genetic Code: Hypothesis of the Coevolution between the Origin of Anticodons and the Evolution of Amino Acids There is a considerable amount of data and suggestions implying a coevolution between the biosynthetic relationships of amino acids and the organisation of the genetic code (for references, see Di Giulio, 1997). If the structuring of the genetic code was also affected by the anticodon–amino acid interactions, then it is reasonable to assume that there might have been a coevolution between the origin of anticodons and the evolution of the biosynthetic pathways of amino acids in such a way that the anticodons came into contact with the amino acids on RNA hairpin structures (Hopfield, 1978; Shimizu, 1995; Di Giulio, 1994, 1996). The only condition which must apparently be satisfied is that, for some reason, the domains of the precursor amino acid codons (Wong, 1975) must have been pre-assigned so as to ensure the contiguity of the amino acids in precursor–product relationships. [This might have been achieved, for instance, by attributing the codons of the genetic code rows to the precursor amino acids
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(Dillon, 1973; Taylor & Coates, 1989; Miseta, 1989) by means of the following simple rule: the first codon base specifies for a certain precursor amino acid (or certain precursor amino acids), even if the anticodons were not yet fully evolved.] Under this condition, it becomes easier to explain why the physicochemical properties of amino acids are reflected in the genetic code together with the observation that the biosynthetic relationships of amino acids are also reflected, and that these two forces did not act independently (Di Giulio, 1996, 1997). Indeed, we need merely postulate that the evolving anticodons still belonging to the precursor amino acids played an active role in selecting the emergent product amino acids. For example, in the biosynthetic transformation Asp : Lys, only when an amino acid (Lys) capable of physicochemical interaction with the anticodons UUU and CUU developed, did the process of exploring the products of Asp terminate as far as these anticodons are concerned. This point of view therefore considers that product amino acids were not at all specified in the initial phase of genetic code evolution and while their selection did indeed depend on anticodons it was largely determined by the history of the biosynthetic relationships between amino acids. Although not entirely free of criticisms, this scheme which thus sees the evolving anticodon playing an active role in the selection of the product amino acid as it affects, addresses and maybe even catalyses the biosynthetic transformations of the precursor amino acid, manages to make the coevolution theory (Wong, 1975) compatible with the stereochemical theory (Woese, 1967) and part of the physicochemical theory (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; Lacey et al., 1992). This hypopthesis which sees the anticodons with an active role in selecting the biosynthetic transformations between amino acids makes one main prediction. As the anticodons under this hypothesis are assumed to have evolved in close contact with the amino acids, possibly on hairpin RNA structures (Hopfield, 1978; Shimizu, 1995; Di Giulio, 1994, 1996), then some anticodons might have been built using amino acids because evolution works on what it has and, more generally, amino acids may be involved in the mechanism by which a tRNA recognises a codon. There are some molecular fossils which seem to support this prediction. Lys modifies a nucleoside present in the first position of the anticodon of a tRNA of Ile (Muramatsu et al., 1988). This modification is essential for the recognition of the codon AUA; moreover aminoacylation specificity is linked to this modified nucleoside in that the unmodified nucleoside accepts Met on the tRNA
.
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while the modified one accepts Ile (Muramatsu et al., 1988). The fact that a tRNA identity determinant is involved indicates that the trait is extremely ancient. Furthermore, all the amino acids involved, as such or as tRNAs, i.e. Lys, Ile and Met, are part of the Asp family in agreement with the coevolution theory (Wong, 1975). Similar considerations hold for a nucleoside modified by Thr (Kasai et al., 1976; Bjork, 1995). This nucleoside, located in position 37 of the tRNAs next to the 3'-side of the anticodon, helps to recognise all the codons that start with A (Nishimura, 1979). Here too, therfore, the modification by Thr takes place on tRNAs belonging mostly to the family of Asp, like Thr, still in agreement with the coevolution theory. Two other nucleosides modified by Gly are described: one is found in position 34 in the anticodons of tRNAs (Yamada et al., 1981; Bjork, 1995) while the location of the other is unknown (Schweizer et al., 1979). Finally, a nucleoside modified by a non-protein amino acid is found in the extra region of tRNAs (Ohashi et al., 1974; Friedman et al., 1974) while the other, modified by norvaline, is perhaps found near the anticodon (Reddy et al., 1992). These are the only known nucleosides modified by amino acids (Limbach et al., 1994). In my opinion these are in clear agreement with the hypothesis mentioned here, above all because they are clearly and almost exclusively located in the tRNA anticodon loops, thus recalling a possible coevolution of anticodons and amino acids.* Time Events in the Origin of the Genetic Code Although not impossible to hypothesise, if there were other, very different forms of genetic code adapted to other functions in early stages of the origin of life, their traces must have been lost probably because of the successive evolution of the code. The discussion here suggests that the organisation of the genetic code emerged in a late phase of the RNA world (Gilbert, 1986), in a phase which gave birth to the ribonucleoprotein world. In this phase tRNA-like * The mean length of a tRNA is 76 nucleotides while the anticodon loop region is seven nucleotides long. Hence the probability of observing a random modification by an amino acid falling in the anticodon loop region, if all the tRNA positions are equally susceptible to such a modification, is 0.092 (7/76). We have observed at least three nucleosides modified by amino acids in the anticodon loop region out of a total of five, as the location of one of them is unknown. Consequently the probability of observing by pure chance at least three out of a total of five modified nucleosides is, using the binomial distribution, only 0.0068
$ 01 n
=s k
%
n k p (1 − p)n − k; p = 0.092, n = 5, k = 3, 4, 5 . k
molecules housing anticodons, or rather their precursors, which were most probably RNA hairpin structures (Hopfield, 1978; Eigen & WinklerOswatitsch, 1981; Bloch et al., 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994) on which biosynthetic transformations of amino acids took place (Wong, 1975), made it possible to link this metabolism to the structure of the genetic code (Wong, 1975; Wachtershauser, 1988; Danchin, 1989; Di Giulio, 1993, 1994). Therefore, I am not in favour of the hypothesis that the regularities detected in the organisation of the genetic code, as regards both the physicochemical properties of amino acids and their biosynthetic relationships (for references, see Szathmary, 1993; Di Giulio, 1997), refer to a pre-tRNA system, as partly supported by Taylor & Coates (1989), but rather that they were achieved on tRNA-like structures. Nor do I support the hypothesis proposed by Szathmary (1993) that the primordial tRNA was the very anticodon that was charged with the amino acid through stereochemical interactions because I believe that the origin of anticodons took place in parallel with the evolution of the biosynthetic pathways of amino acids and all this was achieved on tRNA-like molecules housing evolving anticodons. Moreover, although some similarities can be identified between the hypothesis reported here and that of Szathmary (1993), they differ substantially because the latter suggests that the genetic code originated prior to translation as a pre-adaptation to provide coenzymes ( = anticodons plus amino acids) to ribozymes (Szathmary, 1993) and hence in an early phase of the RNA world. Whereas, the hypothesis referred to here suggests that there was a global coevolution which involved: (i) RNA hairpin structures, which were the precursors of tRNAs (Hopfield, 1978; Eigen & Winkler-Oswatitsch, 1981; Bloch et al., 1985; 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994), housing anticodon-like nucleotides in the stem (Hopfield, 1978; de Duve, 1988; Moller & Janssen, 1992; Mursier-Forsyth & Schimmel, 1993; Schimmel & Henderson, 1994; Di Giulio, 1995; Schimmel, 1995); (ii) the biosynthetic pathways of amino acids occurring on these hairpin structures (Wong, 1975; Wachtershauser, 1988; Danchin, 1989; Di Giulio, 1993, 1994); (iii) a selection by the evolving anticodons on the emerging product amino acids; this phase may have taken place partly in parallel to the direct duplication of hairpin structures to form the complete tRNA molecule (Di Giulio, 1992; 1994; Schimmel, 1995); (iv) a rudimentary protein synthesis achieved initially through simple interactions of
: hairpin structures charged with amino acids (Orgel, 1989; Wong, 1991; Di Giulio, 1994); and finally (v) the organisation of the genetic code proper, i.e. mRNA. Given its complexity, this implies that a late phase of the origin of life witnessed the establishment of the genetic code. To conclude, it is nevertheless not entirely clear why product amino acids physicochemically similar to the anticodons had to be selected in the precursor–product amino acid transformations. The anticodon–amino acid interaction might have been important, for instance, in charging a tRNA, but at this level of complexity a similar interaction is partly unexpected and, because of the hypothesis referred to here, did not help to charge the tRNAs of product amino acids. However, either the system, albeit complex, was evidently not yet sophisticated and thus needed similar recognition interactions, or the recognition between the anticodon and the amino acid was not important per se but because this allowed a generalised lowering of the translation errors by attributing similar codons to similar aminoacids (Woese et al., 1966).
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Reflections on the Origin of the Genetic Code: a Hypothesis M D G International Institute of Genetics and Biophysics, CNR, Via G. Marconi 10, 80125 Naples, Italy (Received on 8 September 1997, Accepted on 29 October 1997)
The origin of the organisation of the genetic code reflects both the biosynthetic relationships between amino acids and the physicochemical interactions between these and anticodons; moreover, these two forces do not act independently. It must therefore be explained why it is simultaneously true that the anticodons of product amino acids were assigned prior to their biosynthetic appearance and that physicochemical correlations must exist between anticodons and amino acids. This gives rise to difficulties of interpretation, even of a more general nature, in the theories that have been proposed to explain the origin of the genetic code; a hypopthesis is thus presented in this manuscript. In particular, the hypothesis suggests that RNA hairpin structures, the ancestors of tRNAs, housing anticodon-like nucleotides in the stem were charged with precursor amino acids. As the precursor amino acids gradually developed into product amino acids, a coevolution came into being between the development of amino acids and that of anticodons, with a concomitant formation of the complete tRNA molecule through direct duplication of the hairpin structures. All this led to the definition of the genetic code organisation. Furthermore, this made it possible for the evolving anticodons to select the emerging product amino acids, which the hypothesis therefore considers to be unspecified in the initial phase of genetic code evolution but which were selected also because of their ability to interact with anticodons. In this way the main obstacle to interpretation is removed. Fossils of these events can now be observed in some amino acid-modified nucleosides specifically located in the tRNA anticodon loops. This is all presented in the framework of a general discussion of the ideas and data in favour of a late origin of the genetic code, as opposed to an early origin in the context of the theories proposed to explain the origin of the genetic code organisation. 7 1998 Academic Press Limited
Introduction A reflection on the time events that led to the origin of the genetic code organisation could provide information useful both for its origin and, more generally, for the origin of life. On the whole, the origin of genetic code organisation might have taken place in an early or a late stage of the origin of life. An early origin of the genetic code organisation has several implications. In view of the considerable complexity of the translation apparatus, the early origin of the genetic code implies that the code was in a very different form from its current one. In other words, the genetic code must have been pre-adapted 0022–5193/98/060191 + 06 $25.00/0/jt970580
to perform other functions which culminated in the genetic code structure proper only with their further evolution. It also seems clear that an early origin might have required some essential aspects of the genetic code to be already in play, such as anticodons or codons per se and amino acids (or some of them). In turn, this seems to imply that a stereochemical interaction between anticodons or codons and amino acids was operative at this stage of genetic code origin. Therefore, an early origin of the genetic code seems to be more consonant with the stereochemical theory of genetic code origin (Woese, 1967) or with part of the physicochemical one (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; 7 1998 Academic Press Limited
192
.
Lacey et al., 1992). More explicitly, if the origin of the genetic code was early this can probably be attributed to stereochemical and/or physicochemical interactions between anticodons or codons and amino acids because these interactions should have been more naturally manifested in such an early stage. Alternatively, a late origin of the genetic code assumes that the code evolved in a system that was already somewhat complex. It is likely that, under the hypothesis of late genetic code origin, the stereochemical hypothesis and part of the physicochemical one lose part of their importance because the system had somehow already abandoned strict physicochemical determinism. On the other hand, the coevolution theory of genetic code origin (Wong, 1975), which is based on the strict parallelism of the biosynthetic relationships between amino acids and the organisation of the genetic code (for references, see Di Giulio, 1997), is more consonant with a late origin of the genetic code as this theory suggests that the organisation of the genetic code was established with the appearance of the biosynthetic pathways of amino acids, which does not intuitively seem to have been a very early event in the origin of life. It was therefore decided to analyse the time events that occurred during the origin of the genetic code, with the aid of the following weak proposition: Since the co-operation of numerous molecules is needed for the achievement of the genetic code, even if it was pre-adapted to perform some other function, it cannot be expected to have an early origin because even the essential elements of the translation apparatus, i.e. aminoacylated tRNAs and mRNAs, imply that the code must have undergone a long evolution which, in turn could mean that the structuring of the genetic code is probably a relatively recent invention of evolution. Therefore, in the present paper ideas and data in favour of the early and the late origin of the genetic code are discussed and the implications and consequences that this has on the theories that have been proposed to explain the origin of genetic code organisation. An Early Origin of the Genetic Code: was There an Ancient Code Based on Anticodon–Amino Acid Interactions? There is a considerable quantity of literature that points to the existence of a relationship between the physicochemical properties of amino acids and the organisation of the genetic code (for references, see Szathmary, 1993; Di Giulio, 1997). The origin of the genetic code might therefore be the direct conse-
quence of an interaction between anticodons or codons and amino acids. In actual fact, in this huge volume of literature there are a few papers that report a correlation between the properties of anticodons and those of amino acids (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; Lacey et al., 1992; Di Giulio, 1996). Other evidence is given by numerous stereochemical models based on interactions between anticodons or codons (or complexes containing them) and amino acids (Pelc & Welton, 1966; Melcher, 1974; Balasubramanian et al., 1980; Hendry et al., 1981; Shimizu, 1982; Yarus, 1991). Moreover, a stereoselective interaction between arginine and codons that codify it in an RNA has been described (Yarus, 1991) and this is therefore, in general, in favour of these models. However, one feels that it is above all the studies conducted on the tRNA identity determinants which seem to provide considerable evidence in favour of the existence of an ancient code positioned in the stem of the hairpin structures (Hopfield, 1978; de Duve, 1988; Moller & Janssen 1992; Mursier-Forsyth & Schimmel, 1993; Schimmel & Henderson, 1994; Di Giulio, 1995; Schimmel, 1995), considered to be the ancestors of the tRNAs (Hopfield, 1978; Eigen & Winkler-Oswatitsch, 1981; Bloch et al., 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994), which imply that the origin of the genetic code might have been based on interactions between anticodons (or codons) and amino acids. In this connection, the observations of Shimizu (1995) also seem to be important as the author finds that hairpin structures housing anticodons in the stem are, in the presence of a dipeptide, specifically charged with the corresponding amino acid. All these and other observations (for references, see Szathmary, 1993; Di Giulio, 1997) suggest that anticodon–amino acid interactions contributed to defining the organisation of the genetic code. It also seems natural to postulate that these interactions were expressed early in the evolution of the code as they are based on what seems to be a strict physicochemical determinism. This leads to the conclusion that there must have been an early phase in which there was a physicochemical correspondence between anticodons (or codons) and amino acids and that, therefore, there might have been a more ancient code based on stereochemical and/or physicochemical interactions. One feels that this conclusion has a very limited validity because it seems to be susceptible to the following two criticisms. (i) In my opinion the origin of the genetic code must first of all reflect aspects of the protein structure because the code evolved in order to build the latter. There are some observations
: suggesting that this might well have been the case (Jurka & Smith, 1987a, b; Di Giulio, 1996). It is also clear that the overlapping of a genetic code reflecting the structural motifs of proteins on the more ancient code based on anticodon–amino acid interactions might have removed many aspects of the latter because the languages of these two codes do not seem to be related. (ii) If there was an ancient genetic code based on anticodon–amino acid interactions before the biosynthetic pathways of amino acids also affected its organisation, what was the result of the successive action of these two forces? Here too it seems that the overlapping of a genetic code reflecting the biosynthetic relationships between amino acids on the one based on anticodon–amino acid interactions might have removed much of the ancient code. Indeed, the biosynthetic relationships between amino acids do not seem to be in the least affected by a pre-existing anticodon–amino acid interaction and these relationships might have even led to the introduction of new amino acids not codified in these interactions. The conclusion reached is that there is some doubt that a more ancient code based on anticodon–amino acid interactions even existed and therefore, in order to take these interactions into account as well, we have to hypothesise that these interactions took place at the same time as the biosynthetic transformations between amino acids. A Late Origin of the Genetic Code: Hypothesis of the Coevolution between the Origin of Anticodons and the Evolution of Amino Acids There is a considerable amount of data and suggestions implying a coevolution between the biosynthetic relationships of amino acids and the organisation of the genetic code (for references, see Di Giulio, 1997). If the structuring of the genetic code was also affected by the anticodon–amino acid interactions, then it is reasonable to assume that there might have been a coevolution between the origin of anticodons and the evolution of the biosynthetic pathways of amino acids in such a way that the anticodons came into contact with the amino acids on RNA hairpin structures (Hopfield, 1978; Shimizu, 1995; Di Giulio, 1994, 1996). The only condition which must apparently be satisfied is that, for some reason, the domains of the precursor amino acid codons (Wong, 1975) must have been pre-assigned so as to ensure the contiguity of the amino acids in precursor–product relationships. [This might have been achieved, for instance, by attributing the codons of the genetic code rows to the precursor amino acids
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(Dillon, 1973; Taylor & Coates, 1989; Miseta, 1989) by means of the following simple rule: the first codon base specifies for a certain precursor amino acid (or certain precursor amino acids), even if the anticodons were not yet fully evolved.] Under this condition, it becomes easier to explain why the physicochemical properties of amino acids are reflected in the genetic code together with the observation that the biosynthetic relationships of amino acids are also reflected, and that these two forces did not act independently (Di Giulio, 1996, 1997). Indeed, we need merely postulate that the evolving anticodons still belonging to the precursor amino acids played an active role in selecting the emergent product amino acids. For example, in the biosynthetic transformation Asp : Lys, only when an amino acid (Lys) capable of physicochemical interaction with the anticodons UUU and CUU developed, did the process of exploring the products of Asp terminate as far as these anticodons are concerned. This point of view therefore considers that product amino acids were not at all specified in the initial phase of genetic code evolution and while their selection did indeed depend on anticodons it was largely determined by the history of the biosynthetic relationships between amino acids. Although not entirely free of criticisms, this scheme which thus sees the evolving anticodon playing an active role in the selection of the product amino acid as it affects, addresses and maybe even catalyses the biosynthetic transformations of the precursor amino acid, manages to make the coevolution theory (Wong, 1975) compatible with the stereochemical theory (Woese, 1967) and part of the physicochemical theory (Weber & Lacey, 1978; Jungck, 1978; Lacey & Mullins, 1983; Lacey et al., 1992). This hypopthesis which sees the anticodons with an active role in selecting the biosynthetic transformations between amino acids makes one main prediction. As the anticodons under this hypothesis are assumed to have evolved in close contact with the amino acids, possibly on hairpin RNA structures (Hopfield, 1978; Shimizu, 1995; Di Giulio, 1994, 1996), then some anticodons might have been built using amino acids because evolution works on what it has and, more generally, amino acids may be involved in the mechanism by which a tRNA recognises a codon. There are some molecular fossils which seem to support this prediction. Lys modifies a nucleoside present in the first position of the anticodon of a tRNA of Ile (Muramatsu et al., 1988). This modification is essential for the recognition of the codon AUA; moreover aminoacylation specificity is linked to this modified nucleoside in that the unmodified nucleoside accepts Met on the tRNA
.
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while the modified one accepts Ile (Muramatsu et al., 1988). The fact that a tRNA identity determinant is involved indicates that the trait is extremely ancient. Furthermore, all the amino acids involved, as such or as tRNAs, i.e. Lys, Ile and Met, are part of the Asp family in agreement with the coevolution theory (Wong, 1975). Similar considerations hold for a nucleoside modified by Thr (Kasai et al., 1976; Bjork, 1995). This nucleoside, located in position 37 of the tRNAs next to the 3'-side of the anticodon, helps to recognise all the codons that start with A (Nishimura, 1979). Here too, therfore, the modification by Thr takes place on tRNAs belonging mostly to the family of Asp, like Thr, still in agreement with the coevolution theory. Two other nucleosides modified by Gly are described: one is found in position 34 in the anticodons of tRNAs (Yamada et al., 1981; Bjork, 1995) while the location of the other is unknown (Schweizer et al., 1979). Finally, a nucleoside modified by a non-protein amino acid is found in the extra region of tRNAs (Ohashi et al., 1974; Friedman et al., 1974) while the other, modified by norvaline, is perhaps found near the anticodon (Reddy et al., 1992). These are the only known nucleosides modified by amino acids (Limbach et al., 1994). In my opinion these are in clear agreement with the hypothesis mentioned here, above all because they are clearly and almost exclusively located in the tRNA anticodon loops, thus recalling a possible coevolution of anticodons and amino acids.* Time Events in the Origin of the Genetic Code Although not impossible to hypothesise, if there were other, very different forms of genetic code adapted to other functions in early stages of the origin of life, their traces must have been lost probably because of the successive evolution of the code. The discussion here suggests that the organisation of the genetic code emerged in a late phase of the RNA world (Gilbert, 1986), in a phase which gave birth to the ribonucleoprotein world. In this phase tRNA-like * The mean length of a tRNA is 76 nucleotides while the anticodon loop region is seven nucleotides long. Hence the probability of observing a random modification by an amino acid falling in the anticodon loop region, if all the tRNA positions are equally susceptible to such a modification, is 0.092 (7/76). We have observed at least three nucleosides modified by amino acids in the anticodon loop region out of a total of five, as the location of one of them is unknown. Consequently the probability of observing by pure chance at least three out of a total of five modified nucleosides is, using the binomial distribution, only 0.0068
$ 01 n
=s k
%
n k p (1 − p)n − k; p = 0.092, n = 5, k = 3, 4, 5 . k
molecules housing anticodons, or rather their precursors, which were most probably RNA hairpin structures (Hopfield, 1978; Eigen & WinklerOswatitsch, 1981; Bloch et al., 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994) on which biosynthetic transformations of amino acids took place (Wong, 1975), made it possible to link this metabolism to the structure of the genetic code (Wong, 1975; Wachtershauser, 1988; Danchin, 1989; Di Giulio, 1993, 1994). Therefore, I am not in favour of the hypothesis that the regularities detected in the organisation of the genetic code, as regards both the physicochemical properties of amino acids and their biosynthetic relationships (for references, see Szathmary, 1993; Di Giulio, 1997), refer to a pre-tRNA system, as partly supported by Taylor & Coates (1989), but rather that they were achieved on tRNA-like structures. Nor do I support the hypothesis proposed by Szathmary (1993) that the primordial tRNA was the very anticodon that was charged with the amino acid through stereochemical interactions because I believe that the origin of anticodons took place in parallel with the evolution of the biosynthetic pathways of amino acids and all this was achieved on tRNA-like molecules housing evolving anticodons. Moreover, although some similarities can be identified between the hypothesis reported here and that of Szathmary (1993), they differ substantially because the latter suggests that the genetic code originated prior to translation as a pre-adaptation to provide coenzymes ( = anticodons plus amino acids) to ribozymes (Szathmary, 1993) and hence in an early phase of the RNA world. Whereas, the hypothesis referred to here suggests that there was a global coevolution which involved: (i) RNA hairpin structures, which were the precursors of tRNAs (Hopfield, 1978; Eigen & Winkler-Oswatitsch, 1981; Bloch et al., 1985; 1985; Schimmel et al., 1993; Moller & Janssen, 1992; Di Giulio, 1992, 1995; Maizels & Weiner, 1994), housing anticodon-like nucleotides in the stem (Hopfield, 1978; de Duve, 1988; Moller & Janssen, 1992; Mursier-Forsyth & Schimmel, 1993; Schimmel & Henderson, 1994; Di Giulio, 1995; Schimmel, 1995); (ii) the biosynthetic pathways of amino acids occurring on these hairpin structures (Wong, 1975; Wachtershauser, 1988; Danchin, 1989; Di Giulio, 1993, 1994); (iii) a selection by the evolving anticodons on the emerging product amino acids; this phase may have taken place partly in parallel to the direct duplication of hairpin structures to form the complete tRNA molecule (Di Giulio, 1992; 1994; Schimmel, 1995); (iv) a rudimentary protein synthesis achieved initially through simple interactions of
: hairpin structures charged with amino acids (Orgel, 1989; Wong, 1991; Di Giulio, 1994); and finally (v) the organisation of the genetic code proper, i.e. mRNA. Given its complexity, this implies that a late phase of the origin of life witnessed the establishment of the genetic code. To conclude, it is nevertheless not entirely clear why product amino acids physicochemically similar to the anticodons had to be selected in the precursor–product amino acid transformations. The anticodon–amino acid interaction might have been important, for instance, in charging a tRNA, but at this level of complexity a similar interaction is partly unexpected and, because of the hypothesis referred to here, did not help to charge the tRNAs of product amino acids. However, either the system, albeit complex, was evidently not yet sophisticated and thus needed similar recognition interactions, or the recognition between the anticodon and the amino acid was not important per se but because this allowed a generalised lowering of the translation errors by attributing similar codons to similar aminoacids (Woese et al., 1966).
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