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On the Dimerization of the Primitive tRNAs: Implications in the Origin of Genetic Code
J. theor. Biol. (2002) 217, 493–498 doi:10.1006/yjtbi.3044, available online at http://www.idealibrary.com on
On the Dimerization of the Primitive tRNAs: Implications in the Origin of Genetic Code Juan A. MartIŁ nez GimeŁ nezw and Rafael TabareŁ s Seisdedos*z wC/oVirgen Del Pilar, 34, 46980 Paterna, Spain and zDepartamento De Medicina, Facultad De Medicina De Valencia, Universidad de Valencia Av. Blasco Iban˜ez 17, 46010 Valencia, Spain (Received on 30 April 2001, Accepted in revised form on 27 March 2002)
RNAs that catalyse their own aminoacylation have been recently selected in vitro. These findings support the notion that the primitive aminoacyl-tRNA synthetases may have been RNAs. In this paper, we propose a structural model for the first aminoacyl-tRNA synthetase consisting of an RNA complex formed between two primitive tRNA molecules through two intermolecular loop-strand interactions, and with implications in the origin of the genetic code. r 2002 Elsevier Science Ltd. All rights reserved.
Introduction Since the beginning of the 1980’s when some cellular RNAs were discovered to have catalytic capacities (Cech et al., 1981; Guerrier-Takada et al., 1983), numerous findings have shown the great functional and structural versatility of catalytic RNAs (Illangasekare et al., 1995; Lohse & Szostak, 1996; Tarasow et al., 1997; Unrau & Bartel, 1998). This makes the RNA world hypothesis more plausible, which establishes that in an earlier stage of life there were only RNA molecules with both catalytic and informational capacity and that later proteins appeared (Gilbert, 1986). Moreover, the definitive proof that ribosome is a ribozyme has been shown (Ban et al., 2000); admittedly one dependent on structural support from protein componentsFgiven that complete deproteina-
*Corresponding author. Tel.: +34-9-638-64168; fax: +349-638-64767 E-mail address: [email protected] (R.T. Seisdedos). 0022-5193/02/$35.00/0
tion destroys peptidyltransferase (Noller et al., 1992). In this molecular context of a possible RNA world, the enigma as to how the code originated remains a mystery although the approach to the matter has changed. The idea is to try and determine how the relationship between RNA and amino acids came about in the absence of proteins, and what was the evolutionary advantage that allowed its selection. The capacity of some selected RNAs in vitro to specifically bind some amino acids has recently been demonstrated (Famulok, 1994; Burgstaller et al., 1995; Majerfeld & Yarus, 1998; Mannironi et al., 2000). This type of specific binding also takes place in the group I self-splicing introns (Yarus, 1988). Although there are reasons for caution (the studied aptamers could be a non-representative sample) (Ellington et al., 2000), several statistical studies of sequences indicate that the specific codons for a particular amino acid in the canonical genetic code occur disproportionately often at RNA r 2002 Elsevier Science Ltd. All rights reserved.
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sites that bind it (Knight & Landweber, 1998; Yarus, 2000). These data support a hypothesis of the origin of code, which states that the codons of genetic code might have originated by the extraction of triplet sequences present within primordial RNA-binding sites (Yarus, 1993, 1998; Knight et al., 1999). On the other hand, the recent findings of two self-aminoacylating ribozymes with amino acid specificity reinforces the notion that the first aminoacyl-tRNA synthetases were made of RNA (Illangasekare & Yarus, 1999; Lee et al., 2000). Moreover, an in vitro evolved catalytic precursor tRNA with self-aminoacylation capacity has been shown (Saito et al., 2001). This finding suggests an RNA-based aminoacylation system capable of charging amino acids onto tRNA-like molecules (Saito et al., 2001). In spite of the importance of this last finding in helping us to understand the evolution of the coding apparatus there are no clues as to how it came about. In other words, what group of biochemical interactions has occurred between amino acids and RNAs was the basis of the genetic code. In this paper, we propose a structural model for the primitive aminoacyl-tRNA synthetase with implications in the origin of genetic code.
anticodon-like triplet located in the 50 -terminal strand), we propose that the primitive tRNAs with identical anticodons could form dimers through two intermolecular loop–strand interactions. On the one hand, the anticodon loop of primitive A tRNA interacts by anti-parallel complementary base pairing with the 30 -terminal strand of the primitive B tRNA, and in other the anticodon loop of the primitive B tRNA interacts by complementary base pairing with the 30 -terminal strand of primitive A tRNA (see Fig. 1). The complementarity of sequences between the anticodon loop and the 30 -terminal strand in the primitive tRNA molecule promote its dimerization. Moreover, other intermolecular forces are involved in dimer stabilization. The divalent metal ions and 20 -hydroxyl groups can have an important role in the non-covalent tRNA–tRNA interactions (between phosphate–ribose backbones
A Possible Dimer of Primitive tRNAS and the Origin of Code The facility for the formation of tRNA dimers through loop–loop interactions of tRNAs with complementary anticodons has been demonstrated by various authors (Grosjean et al., 1973; Eisinger & Gross, 1974). Several studies based on the statistical analysis of tRNA sequences point out the possible presence of codon–anticodon pairs in the accep. tor stem of primitive tRNAs (Moller & Janssen, 1990; Rodin et al., 1996). Moreover, the anticodon-like triplets were mainly concentrated in the 50 acceptor strand of tRNAs (Rodin et al., 1996). On the assumption that some primitive tRNAs, arisen from RNA minihelix, had both a current tRNA-like L-shaped structure and a complementarity of sequence between the anticodon and the 30 -terminal strand (that is, an
Fig. 1. The structure of primitive tRNAs is proposed to have been similar to current tRNAs. (a) Two primitive tRNA molecules with identical anticodons and a codon-like triplet located in the 30 -terminal strand. (b) A dimer formed by primitive tRNA molecules. (c) The dimer where the anticodons occupy the place (locus) of 50 -terminal strand.
THE tRNA DIMERS AND ORIGIN OF CODE
of both monomers) that stabilize the tRNA dimer (Strobel & Doudna, 1997). We propose that the interaction by complementary base pairing involving the anticodon loop of a primitive tRNA and the 30 -terminal strand of other primitive tRNA formed a small duplex structure, which could be considered the structural basis allowing a group of stereochemical interactions with amino acids. We suggest that these stereochemical interactions consisted of the formation of hydrogen bonds between the amino acid sidechain and the bases located in the groove of the 30 -strand-anticodon intermolecular duplex, similar to the one postulated by Yarus (1993). That is, the resulting ‘‘groove’’ formed between the 30 strand and the anticodon loop could allow for specific recognition, through hydrogen bonds, of amino acids, dependent on the anticodon sequence. Moreover, other forces could be involved in the recognition of some amino acids, as the electrostatic interactions. The type of 30 strand:anticodon–amino acid interaction may have been a progenitor of the genetic code. These interactions would have largely determined the correspondence between certain RNA-sequence tags (RNA sites) and various amino acids. Other structural elements close to the anticodon-30 strand duplex could be involved in the stabilization of the interaction between activated amino acid and duplex, as some non-paired nucleotides of 30 -end. We propose that this RNA dimer also could catalyse their own specific aminoacylation. The interaction between the anticodon and the 30 terminal strand in the molecular context of dimer could generate (form) a structure capable of recognizing and specifically binding an amino acid close to the 30 -end of one tRNA of dimer, positioning the activated amino acid for nucleophilic attack by the 30 OH of the 30 -terminal ribose to yield an aminoacyl-tRNA. Moreover, we suggest that one metal ion could activate the terminal 30 -hydroxyl (that is, a role in forming the ribose nucleophile). The intermolecular interaction between the anticodon loop and the 30 strand could produce one metal-binding site close to the 30 -terminal ribose. In fact, the likely locations for binding metal ions are sites with a high concentration of phosphate ions, as the junction of loop and stem. Moreover, various
495
metal ions have been found in the crystal structure of current tRNAs (Pan et al., 1993). RNA enzymes usually depend on a catalytic metal (Pyle, 1993). We propose that the tRNA dimer could have used as activated amino acid, the aminoacylthioesters. The aminoacyl-thioesters do not suffer from hydrolytic lability, unlike aminoacyl-adenylates. These molecules could have been the prebiotically available components of the ‘‘thioester world’’ (De Duve, 1991), which might have co-existed with the RNA world. Moreover, it has been shown that one tRNA with a catalytic additional domain accepts the thioesters as the substrate (Saito et al., 2001), which supports the idea that such thioesters could have been used as competent aminoacyl donors in the early RNA-based aminoacylation system. The tRNA dimer had two catalytic centers located in the sites of intermolecular loop–strand interactions. The dissociation of dimer occurs after aminoacylation of both monomers, producing two aminoacyl-tRNAs. In this model, only the dimer is proposed to have been able to bind the amino acid and to catalyse their own aminoacylation, unlike the single tRNA. That is, a single tRNA cannot bind and catalyse its own aminoacylation. Moreover, not all the primitive tRNAs could form dimers, only those where a relationship of complementarity between the anticodon triplet and the 30 terminal strand was present. The RNA dimer allowed that the parts of the tRNA molecule, where the majority of identity determinants are located separately (anticodon and acceptor stem), are to be arranged together and to interact all at once with the amino acid when assignment begins to be established and thus intervening in this process. This type of double interaction allows us to understand how a relationship could have been established between an amino acid and its corresponding codon. With the appearance of protein synthetases the requirement of sequence complementarity could be more relaxed or even the presence of one element of dimer could be rendered unnecessary. In brief, long coevolution of tRNAs and protein synthetases did not conserve the relationship of complementarity between
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anticodon and the 30 end in its initial form (Rodin et al., 1996). Discussion On the one hand, the capacity shown by the simple RNA structures bind several amino acids with notable specificity (Famulok, 1994; Burgstaller et al., 1995; Majerfeld & Yarus, 1998; Mannironi et al., 2000) and on the other hand the selected self-aminoacylating ribozymes (Illangasekare & Yarus, 1999; Lee et al., 2000) reinforce the notion that a determined group of specific interactions arose between isolated amino acids and certain RNA structures during the RNA stage of life, which was possibly the embryo of the genetic code. However, the question is to know what this specific interaction consists of and what type of RNA structure could have been the base material for it. It has been postulated that tRNA or tRNAlike molecules are among the oldest RNAs surviving from the RNA world (Schimmel & . 1997). A phylogenetic analysis suggests that Soll, the tRNA molecules were already in existence when the proto-translation system started to evolve (De Pouplana et al., 1998). Moreover, it has been recently demonstrated that an ‘‘in vitro’’ evolved precursor tRNA consisting of two domains, a catalytic 50 -leader sequence and an aminoacyl-acceptor tRNA, can catalyse its self-aminoacylation with remarkable amino acid selectivity (Saito et al., 2001). This finding suggests a possible evolutionary pathway for tRNA aminoacylation (Saito et al., 2001). However, it remains a mystery as to how a relationship between amino acids and the tRNA sequences relevant to the genetic code (anticodon and acceptor stem) could have been established in the origins of life. Our model could provide a possible explanation to this question. In this paper, we propose a structural model for the first aminoacyl-tRNA synthetase consisting of a complex formed between two primitive tRNA molecules through two intermolecular loop–strand interactions. In this model, the structure responsible for amino acid specificity
in the tRNA aminoacylation is an intermolecular duplex formed between anticodon loop and 30 -strand, that is, the tRNA sequences that could be relevant to the origin of the code. We propose that some of the acceptor stemspecific, current identity elements could have taken part in the anticodon/30 -strand duplex of the primitive dimer. At the molecular level, in the protein–DNA world, we know that the determinants of the tRNA identity are mainly located in the anticodon loop and the acceptor stem (Giege et al., 1998). Both parts are well distanced from each other in the molecule and the presence of proteins is necessary in order to read these RNA determinants in an amino acid. Major determinants are conserved in the evolution. It has also been suggested that the minihelix with acceptor stem was determinant in establishing the relationship between amino acids and RNAs, operational RNA code (Schimmel et al., 1993). However, the sequence diversity in the current acceptor region seems insufficient to discriminate the 20 natural amino acids (Saks & Sampson, 1995; Rodin et al., 1996). This problem may have been overcome with the emergence of a tRNA-like molecule having an anticodon loop in the moment of assignment of amino acids (Saks & Sampson, 1995). This model offers an explanation for the transition from primitive ribozymes to modern synthetases. It is extensively believed that during the RNA world arose the first-coded polypeptides. Some of them would be able to recognize and bind with the dimers of primitive tRNAs. On binding, the RNA dimer then became more stable and the catalytic capacity therefore improved. The need for complementary base pairing (tRNA–tRNA interactions) would be largely reduced, and the catalytic capacity would then become due to the protein (progression identical to that proposed by White, 1976). Presumably, the selective pressures to maintain these tRNA–tRNA interactions were reduced as a function of the increased role of the peptide/ protein component that eventually became the synthetase. Finally, the protein was able to aminoacylate a single tRNA in the absence of the other. In brief, the primitive active center completely disappeared on eliminating a tRNA.
497
THE tRNA DIMERS AND ORIGIN OF CODE
This idea that a stabilizing peptide could have eventually taken over the role of one of the monomers of the primitive tRNA dimer and appropriate the catalytic function, brings up the possibility of molecular mimicry of catalytic RNAs by the proteins that usurped their role during the evolution of the tRNA aminoacylation system. In fact, genetic translation contains a protein component that mimic tRNA, the elongation factor EF-G (Nissen et al., 1995); which suggests that the primeval translocase could have been made of RNA–tRNA (Wilson & Noller, 1998). Recently, it has been found that a peptide determinant in polypeptide release factors equivalent to the anticodon of tRNA (Ito et al., 2000). Thus, it is possible that, initially, the tRNA or tRNA-like structures could have played an essential role in the various steps of the process of primitive protein synthesis. The molecular RNA mimicry by protein could have been an important step in the transition from the RNA world to the ribonucleo-protein world. Other possible mechanisms have been suggested for the passing from the RNA world to ribonucleo-protein world (Mart!ınez Gime! nez et al., 1998). It is possible that an important step in the transition from tRNA dimer to protein synthetase could have been the mimicry of a part of anticodon-30 -strand duplex by the protein. This could explain that the minihelices recapitulating acceptor branches of tRNAs in which the major identity elements are found in the anticodon loop can be also charged by their synthetases (Giege! et al., 1998). On the other hand, the striking difference between the protein synthetase and tRNA dimer is the amino acid activation. It is possible that some stabilizing peptides capable of binding an ATP molecule and catalysing the amino acid activation could have evolved. The quaternary assembly proposed may prove to be a prominent feature of RNA functional evolution. This theoretical, structural model proposed can be researched experimentally. tRNAs with anticodon-like triplets in the 50 terminal strand (that is, with codon-like triplets in the 30 -terminal strand) would have to be used in the experiment in order to demonstrate its capacity to form catalytic dimers.
The tRNA is a ubiquitous molecule which is involved in many more reactions than genetic translation. The wide variety of uses for tRNA in cell metabolism may indicate that in the past, tRNA or tRNA-like molecules did assume various enzymatic roles which in the course of evolution were usurped by the proteins that . 1993). today interact with RNA (Soll, We propose that tRNA dimers could have performed various catalytic activities in the RNA world. We suggest that a ‘‘world’’ formed by the tRNA dimers could have existed as an important part within the RNA world.
REFERENCES Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steiz, T. A. (2000). The complete atomic structure of the large ( resolution. Science 289, ribosomal subunit at 2.4 A 905–920. Burgstaller, P., Kochollan, M. & Famulok, M. (1995). Structural probing and damage selection of citrulline- and arginine-specific RNA aptamers identify base positions required for binding. Nucl. Acids Res. 23, 4769–4776. Cech, T. R., Zaug, A. J. & Grabowski, P. J. (1981). In vitro splicing of the ribosomal RNA precursor of Tetrahymena; involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27, 487–496. De Duve, C. (1991). Blueprint for a Cell: The Nature and Origin of Life. Burlington, NC: N. Patterson Publishers. De Pouplana, L. R., Turner, R. J., Steer, B. A. & Schimmel, P. (1998). Genetic code origins: tRNAs older than their synthetases? Proc. Natl Acad. Sci. U.S.A. 95, 11 295–11 300. Ellington, A. D., Khrapov, M. & Shaw, C. A. (2000). The scene of a frozen accident. RNA 6, 485–498. Eisinger, J. & Gross, N. (1974). The anticodon–anticodon complex. J. Mol. Biol. 88, 165–174. Famulok, M. (1994). Molecular recognition of amino acids by RNA-aptamers: an L-citruline binding RNA motif and its evolution into an L-arginine binder. J. Am. Chem. Sos. 116, 1698–1706. Giege, R., Sissler, M. & Florentz, C. (1998). Universal rules and idiosyncratic features in tRNA identity. Nucl. Acids Res. 26, 5017–5035. Gilbert, W. (1986). The RNA world. Nature 319, 618. Grosjean, H., Takada, C. & Petre, P. (1973). Complex formation between tRNAs with complementary anticodons; use of matrix bound tRNA. Biochem. Biophys. Res. Commun. 53, 882–893. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. & Altman, S. (1983). The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849–857. Illangasekare, M. & Yarus, M. (1999). Specific, rapid synthesis of Phe-RNA by RNA. Proc. Natl Acad. Sci. U.S.A. 96, 5470–5475.
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Illangasekare, M., Sanchez, G., Nickles, T. & Yarus, M. (1995). Aminoacyl-RNA synthesis catalyzed by an RNA. Science 267, 643–647. Ito, K., Uno, M. & Nakamura, Y. (2000). A tripeptide ‘‘anticodon’’ deciphers stop codons in messenger RNA. Nature 403, 680–684. Knight, R. D. & Landweber, L. F. (1998). Rhyme or reason: RNA–arginine interactions and the genetic code. Chem. Biol. 5, R215-R220. Knight, R. D., Freeland, S. J. & Landweber, L. F. (1999). Selection, history and chemistry: the tree faces of the genetic code. Trends Biochem. Sci. 24, 241–247. Lee, N., Bessho, Y., Wei, K., Szostak, J. W. & Suga, H. (2000). Ribozyme-catalyzed tRNA aminoacylation. Nat. Struct. Biol. 7, 28–33. Lohse, P. A. & Szostak, J. W. (1996). Ribozyme-catalysed amino-acid transfer reactions. Nature 381, 442–444. Majerfeld, I. & Yarus, M. (1998). Isoleucine: RNA sites with associated coding sequences. RNA 4, 471–478. Mannironi, C., Scherch, C., Fruscoloni, P. & Tocchini-Valentini, G. P. (2000). Molecular recognition of amino acids by RNA aptamers: the evolution into a L-tyrosine binder of a dopamine binding motif. RNA 6, 520–527. Martinez Gimenez, J. A., Saez, G. T. & Tabares Seisdedos, R. (1998). On the function of modified nucleosides in the RNA world. J. theor. Biol. 194, 485–490. Moº ller, W. & Janssen, G. M. C. (1990). Transfer RNAs for primordial amino acids contain remnants of a primitive code at posiiton 3 to 5. Biochimie 72, 361–368. Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B. F. & Nyborg, J. (1995). Crystal structure of the ternary complex of PhetRNA(Phe), EF-Tu, and a GTP analog. Science 270, 1464–1472. Noller, H. F., Hoffarth, V. & Zimniak, L. (1992). Unusual resistence of peptidyltransferase to protein extraction methods. Science 256, 1416–1419. Pan, T., Long, D. M. & Uhlenbeck, O. C. (1993). Divalent metal ions in RNA folding and catalysis. In: The RNA World (Gesteland, R. & Atkins, J., eds), pp. 271–302. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Pyle, A. M. (1993). Ribozymes: a distinct class of metalloenzymes. Science 261, 709–714. Rodin, S., Rodin, A. & Ohno, S. (1996). The presence of codon–anticodon pairs in the acceptor stem of tRNAs. Proc. Natl Acad. Sci. U.S.A. 93, 4537–4542. Saito, H., Kourouklis, D. & Suga, H. (2001). An in vitro evolved precursor tRNA with aminoacylation activity. EMBO J. 20, 1797–1806. Saks, M. E. & Sampson, J. R. (1995). Evolution of tRNA recognition systems and tRNA gene sequences. J. Mol. Evol. 40, 509–518. Schimmel, P. & Soº ll, D. (1997). When protein engineering confronts the tRNA world. Proc. Natl Acad. Sci. U.S.A. 94, 10 007–10 009. Schimmel, P., Giege, R., Moras, D. & Yokoyama, S. (1993). An operational RNA code for amino acids and possible relationship to genetic code. Proc. Natl Acad. Sci. U.S.A. 90, 8763–8768. Soº ll, D. (1993). Transfer RNA: an RNA for all seasons. In: The RNA World (Gesteland, R. F. & Atkins, J. F., eds), pp. 157–177. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Strobel, S. A. & Doudna, J. A. (1997). RNA seeing double: close-packing of helices in RNA tertiary structure. Trends Biochem. Sci. 22, 262–266. Tarasow, T. M., Tarasow, S. L. & Eaton, B. E. (1997). RNA-catalyzed carbon–carbon bond formation. Nature 389, 54–57. Unrau, P. J. & Bartel, D. P. (1998). RNA-catalysed nucleotide synthesis. Nature 395, 260–263. White, III H. B. (1976). Coenzymes as fossils of an earlier metabolic state. J. Mol. Evol. 7, 101–104. Wilson, K. S. & Noller, H. F. (1998). Molecular movement inside the translational engine. Cell 92, 337–349. Yarus, M. (1988). A specific amino acid binding site composed of RNA. Science 240, 1751–1758. Yarus, M. (1993). An RNA–amino acid affinity. In: The RNA World (Gesteland, R. F. & Atkins, J. F. eds), pp. 205–217. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Yarus, M. (1998). Amino acids as RNA ligands: a directRNA-template theory for the code’s origin. J. Mol. Evol. 47, 109–117. Yarus, M. (2000). RNA ligand chemistry; a testable source for the genetic code. RNA 6, 475–484.
On the Dimerization of the Primitive tRNAs: Implications in the Origin of Genetic Code Juan A. MartIŁ nez GimeŁ nezw and Rafael TabareŁ s Seisdedos*z wC/oVirgen Del Pilar, 34, 46980 Paterna, Spain and zDepartamento De Medicina, Facultad De Medicina De Valencia, Universidad de Valencia Av. Blasco Iban˜ez 17, 46010 Valencia, Spain (Received on 30 April 2001, Accepted in revised form on 27 March 2002)
RNAs that catalyse their own aminoacylation have been recently selected in vitro. These findings support the notion that the primitive aminoacyl-tRNA synthetases may have been RNAs. In this paper, we propose a structural model for the first aminoacyl-tRNA synthetase consisting of an RNA complex formed between two primitive tRNA molecules through two intermolecular loop-strand interactions, and with implications in the origin of the genetic code. r 2002 Elsevier Science Ltd. All rights reserved.
Introduction Since the beginning of the 1980’s when some cellular RNAs were discovered to have catalytic capacities (Cech et al., 1981; Guerrier-Takada et al., 1983), numerous findings have shown the great functional and structural versatility of catalytic RNAs (Illangasekare et al., 1995; Lohse & Szostak, 1996; Tarasow et al., 1997; Unrau & Bartel, 1998). This makes the RNA world hypothesis more plausible, which establishes that in an earlier stage of life there were only RNA molecules with both catalytic and informational capacity and that later proteins appeared (Gilbert, 1986). Moreover, the definitive proof that ribosome is a ribozyme has been shown (Ban et al., 2000); admittedly one dependent on structural support from protein componentsFgiven that complete deproteina-
*Corresponding author. Tel.: +34-9-638-64168; fax: +349-638-64767 E-mail address: [email protected] (R.T. Seisdedos). 0022-5193/02/$35.00/0
tion destroys peptidyltransferase (Noller et al., 1992). In this molecular context of a possible RNA world, the enigma as to how the code originated remains a mystery although the approach to the matter has changed. The idea is to try and determine how the relationship between RNA and amino acids came about in the absence of proteins, and what was the evolutionary advantage that allowed its selection. The capacity of some selected RNAs in vitro to specifically bind some amino acids has recently been demonstrated (Famulok, 1994; Burgstaller et al., 1995; Majerfeld & Yarus, 1998; Mannironi et al., 2000). This type of specific binding also takes place in the group I self-splicing introns (Yarus, 1988). Although there are reasons for caution (the studied aptamers could be a non-representative sample) (Ellington et al., 2000), several statistical studies of sequences indicate that the specific codons for a particular amino acid in the canonical genetic code occur disproportionately often at RNA r 2002 Elsevier Science Ltd. All rights reserved.
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sites that bind it (Knight & Landweber, 1998; Yarus, 2000). These data support a hypothesis of the origin of code, which states that the codons of genetic code might have originated by the extraction of triplet sequences present within primordial RNA-binding sites (Yarus, 1993, 1998; Knight et al., 1999). On the other hand, the recent findings of two self-aminoacylating ribozymes with amino acid specificity reinforces the notion that the first aminoacyl-tRNA synthetases were made of RNA (Illangasekare & Yarus, 1999; Lee et al., 2000). Moreover, an in vitro evolved catalytic precursor tRNA with self-aminoacylation capacity has been shown (Saito et al., 2001). This finding suggests an RNA-based aminoacylation system capable of charging amino acids onto tRNA-like molecules (Saito et al., 2001). In spite of the importance of this last finding in helping us to understand the evolution of the coding apparatus there are no clues as to how it came about. In other words, what group of biochemical interactions has occurred between amino acids and RNAs was the basis of the genetic code. In this paper, we propose a structural model for the primitive aminoacyl-tRNA synthetase with implications in the origin of genetic code.
anticodon-like triplet located in the 50 -terminal strand), we propose that the primitive tRNAs with identical anticodons could form dimers through two intermolecular loop–strand interactions. On the one hand, the anticodon loop of primitive A tRNA interacts by anti-parallel complementary base pairing with the 30 -terminal strand of the primitive B tRNA, and in other the anticodon loop of the primitive B tRNA interacts by complementary base pairing with the 30 -terminal strand of primitive A tRNA (see Fig. 1). The complementarity of sequences between the anticodon loop and the 30 -terminal strand in the primitive tRNA molecule promote its dimerization. Moreover, other intermolecular forces are involved in dimer stabilization. The divalent metal ions and 20 -hydroxyl groups can have an important role in the non-covalent tRNA–tRNA interactions (between phosphate–ribose backbones
A Possible Dimer of Primitive tRNAS and the Origin of Code The facility for the formation of tRNA dimers through loop–loop interactions of tRNAs with complementary anticodons has been demonstrated by various authors (Grosjean et al., 1973; Eisinger & Gross, 1974). Several studies based on the statistical analysis of tRNA sequences point out the possible presence of codon–anticodon pairs in the accep. tor stem of primitive tRNAs (Moller & Janssen, 1990; Rodin et al., 1996). Moreover, the anticodon-like triplets were mainly concentrated in the 50 acceptor strand of tRNAs (Rodin et al., 1996). On the assumption that some primitive tRNAs, arisen from RNA minihelix, had both a current tRNA-like L-shaped structure and a complementarity of sequence between the anticodon and the 30 -terminal strand (that is, an
Fig. 1. The structure of primitive tRNAs is proposed to have been similar to current tRNAs. (a) Two primitive tRNA molecules with identical anticodons and a codon-like triplet located in the 30 -terminal strand. (b) A dimer formed by primitive tRNA molecules. (c) The dimer where the anticodons occupy the place (locus) of 50 -terminal strand.
THE tRNA DIMERS AND ORIGIN OF CODE
of both monomers) that stabilize the tRNA dimer (Strobel & Doudna, 1997). We propose that the interaction by complementary base pairing involving the anticodon loop of a primitive tRNA and the 30 -terminal strand of other primitive tRNA formed a small duplex structure, which could be considered the structural basis allowing a group of stereochemical interactions with amino acids. We suggest that these stereochemical interactions consisted of the formation of hydrogen bonds between the amino acid sidechain and the bases located in the groove of the 30 -strand-anticodon intermolecular duplex, similar to the one postulated by Yarus (1993). That is, the resulting ‘‘groove’’ formed between the 30 strand and the anticodon loop could allow for specific recognition, through hydrogen bonds, of amino acids, dependent on the anticodon sequence. Moreover, other forces could be involved in the recognition of some amino acids, as the electrostatic interactions. The type of 30 strand:anticodon–amino acid interaction may have been a progenitor of the genetic code. These interactions would have largely determined the correspondence between certain RNA-sequence tags (RNA sites) and various amino acids. Other structural elements close to the anticodon-30 strand duplex could be involved in the stabilization of the interaction between activated amino acid and duplex, as some non-paired nucleotides of 30 -end. We propose that this RNA dimer also could catalyse their own specific aminoacylation. The interaction between the anticodon and the 30 terminal strand in the molecular context of dimer could generate (form) a structure capable of recognizing and specifically binding an amino acid close to the 30 -end of one tRNA of dimer, positioning the activated amino acid for nucleophilic attack by the 30 OH of the 30 -terminal ribose to yield an aminoacyl-tRNA. Moreover, we suggest that one metal ion could activate the terminal 30 -hydroxyl (that is, a role in forming the ribose nucleophile). The intermolecular interaction between the anticodon loop and the 30 strand could produce one metal-binding site close to the 30 -terminal ribose. In fact, the likely locations for binding metal ions are sites with a high concentration of phosphate ions, as the junction of loop and stem. Moreover, various
495
metal ions have been found in the crystal structure of current tRNAs (Pan et al., 1993). RNA enzymes usually depend on a catalytic metal (Pyle, 1993). We propose that the tRNA dimer could have used as activated amino acid, the aminoacylthioesters. The aminoacyl-thioesters do not suffer from hydrolytic lability, unlike aminoacyl-adenylates. These molecules could have been the prebiotically available components of the ‘‘thioester world’’ (De Duve, 1991), which might have co-existed with the RNA world. Moreover, it has been shown that one tRNA with a catalytic additional domain accepts the thioesters as the substrate (Saito et al., 2001), which supports the idea that such thioesters could have been used as competent aminoacyl donors in the early RNA-based aminoacylation system. The tRNA dimer had two catalytic centers located in the sites of intermolecular loop–strand interactions. The dissociation of dimer occurs after aminoacylation of both monomers, producing two aminoacyl-tRNAs. In this model, only the dimer is proposed to have been able to bind the amino acid and to catalyse their own aminoacylation, unlike the single tRNA. That is, a single tRNA cannot bind and catalyse its own aminoacylation. Moreover, not all the primitive tRNAs could form dimers, only those where a relationship of complementarity between the anticodon triplet and the 30 terminal strand was present. The RNA dimer allowed that the parts of the tRNA molecule, where the majority of identity determinants are located separately (anticodon and acceptor stem), are to be arranged together and to interact all at once with the amino acid when assignment begins to be established and thus intervening in this process. This type of double interaction allows us to understand how a relationship could have been established between an amino acid and its corresponding codon. With the appearance of protein synthetases the requirement of sequence complementarity could be more relaxed or even the presence of one element of dimer could be rendered unnecessary. In brief, long coevolution of tRNAs and protein synthetases did not conserve the relationship of complementarity between
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anticodon and the 30 end in its initial form (Rodin et al., 1996). Discussion On the one hand, the capacity shown by the simple RNA structures bind several amino acids with notable specificity (Famulok, 1994; Burgstaller et al., 1995; Majerfeld & Yarus, 1998; Mannironi et al., 2000) and on the other hand the selected self-aminoacylating ribozymes (Illangasekare & Yarus, 1999; Lee et al., 2000) reinforce the notion that a determined group of specific interactions arose between isolated amino acids and certain RNA structures during the RNA stage of life, which was possibly the embryo of the genetic code. However, the question is to know what this specific interaction consists of and what type of RNA structure could have been the base material for it. It has been postulated that tRNA or tRNAlike molecules are among the oldest RNAs surviving from the RNA world (Schimmel & . 1997). A phylogenetic analysis suggests that Soll, the tRNA molecules were already in existence when the proto-translation system started to evolve (De Pouplana et al., 1998). Moreover, it has been recently demonstrated that an ‘‘in vitro’’ evolved precursor tRNA consisting of two domains, a catalytic 50 -leader sequence and an aminoacyl-acceptor tRNA, can catalyse its self-aminoacylation with remarkable amino acid selectivity (Saito et al., 2001). This finding suggests a possible evolutionary pathway for tRNA aminoacylation (Saito et al., 2001). However, it remains a mystery as to how a relationship between amino acids and the tRNA sequences relevant to the genetic code (anticodon and acceptor stem) could have been established in the origins of life. Our model could provide a possible explanation to this question. In this paper, we propose a structural model for the first aminoacyl-tRNA synthetase consisting of a complex formed between two primitive tRNA molecules through two intermolecular loop–strand interactions. In this model, the structure responsible for amino acid specificity
in the tRNA aminoacylation is an intermolecular duplex formed between anticodon loop and 30 -strand, that is, the tRNA sequences that could be relevant to the origin of the code. We propose that some of the acceptor stemspecific, current identity elements could have taken part in the anticodon/30 -strand duplex of the primitive dimer. At the molecular level, in the protein–DNA world, we know that the determinants of the tRNA identity are mainly located in the anticodon loop and the acceptor stem (Giege et al., 1998). Both parts are well distanced from each other in the molecule and the presence of proteins is necessary in order to read these RNA determinants in an amino acid. Major determinants are conserved in the evolution. It has also been suggested that the minihelix with acceptor stem was determinant in establishing the relationship between amino acids and RNAs, operational RNA code (Schimmel et al., 1993). However, the sequence diversity in the current acceptor region seems insufficient to discriminate the 20 natural amino acids (Saks & Sampson, 1995; Rodin et al., 1996). This problem may have been overcome with the emergence of a tRNA-like molecule having an anticodon loop in the moment of assignment of amino acids (Saks & Sampson, 1995). This model offers an explanation for the transition from primitive ribozymes to modern synthetases. It is extensively believed that during the RNA world arose the first-coded polypeptides. Some of them would be able to recognize and bind with the dimers of primitive tRNAs. On binding, the RNA dimer then became more stable and the catalytic capacity therefore improved. The need for complementary base pairing (tRNA–tRNA interactions) would be largely reduced, and the catalytic capacity would then become due to the protein (progression identical to that proposed by White, 1976). Presumably, the selective pressures to maintain these tRNA–tRNA interactions were reduced as a function of the increased role of the peptide/ protein component that eventually became the synthetase. Finally, the protein was able to aminoacylate a single tRNA in the absence of the other. In brief, the primitive active center completely disappeared on eliminating a tRNA.
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THE tRNA DIMERS AND ORIGIN OF CODE
This idea that a stabilizing peptide could have eventually taken over the role of one of the monomers of the primitive tRNA dimer and appropriate the catalytic function, brings up the possibility of molecular mimicry of catalytic RNAs by the proteins that usurped their role during the evolution of the tRNA aminoacylation system. In fact, genetic translation contains a protein component that mimic tRNA, the elongation factor EF-G (Nissen et al., 1995); which suggests that the primeval translocase could have been made of RNA–tRNA (Wilson & Noller, 1998). Recently, it has been found that a peptide determinant in polypeptide release factors equivalent to the anticodon of tRNA (Ito et al., 2000). Thus, it is possible that, initially, the tRNA or tRNA-like structures could have played an essential role in the various steps of the process of primitive protein synthesis. The molecular RNA mimicry by protein could have been an important step in the transition from the RNA world to the ribonucleo-protein world. Other possible mechanisms have been suggested for the passing from the RNA world to ribonucleo-protein world (Mart!ınez Gime! nez et al., 1998). It is possible that an important step in the transition from tRNA dimer to protein synthetase could have been the mimicry of a part of anticodon-30 -strand duplex by the protein. This could explain that the minihelices recapitulating acceptor branches of tRNAs in which the major identity elements are found in the anticodon loop can be also charged by their synthetases (Giege! et al., 1998). On the other hand, the striking difference between the protein synthetase and tRNA dimer is the amino acid activation. It is possible that some stabilizing peptides capable of binding an ATP molecule and catalysing the amino acid activation could have evolved. The quaternary assembly proposed may prove to be a prominent feature of RNA functional evolution. This theoretical, structural model proposed can be researched experimentally. tRNAs with anticodon-like triplets in the 50 terminal strand (that is, with codon-like triplets in the 30 -terminal strand) would have to be used in the experiment in order to demonstrate its capacity to form catalytic dimers.
The tRNA is a ubiquitous molecule which is involved in many more reactions than genetic translation. The wide variety of uses for tRNA in cell metabolism may indicate that in the past, tRNA or tRNA-like molecules did assume various enzymatic roles which in the course of evolution were usurped by the proteins that . 1993). today interact with RNA (Soll, We propose that tRNA dimers could have performed various catalytic activities in the RNA world. We suggest that a ‘‘world’’ formed by the tRNA dimers could have existed as an important part within the RNA world.
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