RNA-directed amino acid homochirality

^a Department of Biochemistry and Molecular Biology, The George Washington University School of Medicine and Health Sciences, Washington, D.C. 20037, USA

	ABSTRACT

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The phenomenon of L-amino acid homochirality was analyzed on the basis that protein synthesis evolved in an environment in which ribose nucleic acids preceded proteins, so that selection of L-amino acids may have arisen as a consequence of the properties of the RNA molecule. Aminoacylation of RNA is the primary mechanism for selection of amino acids for protein synthesis, and models of this reaction with both D- and L-amino acids have been constructed. It was confirmed, as observed by others, that the aminoacylation of RNA by amino acids in free solution is not predictably stereoselective. However, when the RNA molecule is constrained on a surface (mimicking prebiotic surface monolayers), it becomes automatically selective for the L-enantiomers. Conversely, L-ribose RNA would have been selective for the D-isomers. Only the 2' aminoacylation of surface-bound RNA would have been stereoselective. This finding may explain the origin of the redundant 2' aminoacylation still undergone by a majority of today's amino acids before conversion to the 3' species required for protein synthesis. It is concluded that L-amino acid homochirality was predetermined by the prior evolution of D-ribose RNA and probably was chirally directed by the orientation of early RNA molecules in surface monolayers.—Bailey, J. M. RNA-directed amino acid homochirality. FASEB J. 12, 503–507 (1998)

Key Words: aminoacylation • protein synthesis • biopolymer • chiroselection • aminoacyl-tRNA synthase translation • L-amino acids • evolution • codon • stereoisomerism

	INTRODUCTION

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THE TWENTY OR SO amino acids found in living organisms occur almost exclusively as L-enantiomers. The origins of this pervasive homochirality have never been explained satisfactorily. Because there is no obvious biochemical reason for choosing L-amino acids over the D-forms, numerous physical and biochemical mechanisms have been invoked to explain the phenomenon (1). Physical mechanisms for the generation of chirality have been proposed based on the discovery of parity violations in elementary particle interactions. In the case of amino acids, parity violation would generate an excess of L-enantiomers over the D-forms. However, in 1 mol of racemic amino acids, the excess of L-enantiomer would amount to only about 1 million molecules, or one part in 10¹⁸ (2). It has also been proposed recently that enantiomeric enrichment may have occurred in an interstellar environment by a process involving the polarized synchrotron radiation around neutron stars, resulting in the early Earth being seeded with a preordained molecular homochirality (3). Other reasoning has led to speculation that homochirality was a necessary condition for life and that selection of structures containing only the L-amino acids occurred by chance. Miller and Orgel (1), however, have suggested that isotactic polymers (containing monomeric units that are all of the same handedness) may have formed randomly, yielding equal numbers of D- and L-based polymers. In support of this idea, Brack and Spach (4, 5) have demonstrated that when the residue composition of the protein -helix departs from the racemic mixture, continued growth of the helix favors one-handedness. Similarly, the formation of ß-sheets arises from the association of chain segments containing at least seven residues of the same chirality (4, 5). This mechanism, although demonstrating how enantiomeric segregation may occur, does not explain the preference for one enantiomer over the other. An analogous process for chiroselection in an oligonucleotide system has recently been described by Bolli et al. (6), in which a chiroselective self-assembly of base sequences by oligomerization of tetranucleotide cyclophosphates was observed.

Even though plausible physical and chemical mechanisms have been proposed to explain enantio-meric enrichment of amino acids and other precursors of biopolymers, the phenomenon of amino acid homochirality must also be evaluated in light of the evidence that life evolved in an environment in which ribose nucleic acids were the dominant molecules. In the early stages, both genetic and catalytic functions probably were carried out by RNA (7, 8), and the need for proteins probably evolved later. If RNA preceded proteins, selection of the L-enantiomers of the amino acids may well have arisen as a consequence of the chirality of the RNA molecule itself. The only chiral moiety in RNA is D-ribose, since none of the purine or pyrimidine bases possess a chiral center. The key step in the selection of amino acids for protein synthesis is aminoacylation of the D-ribose group. If this reaction were to display an abiotic selectivity for the L-enantiomer of one amino acid, then it would most likely display the same selectivity toward the L-enantiomers of all the other amino acids. Profy and Usher (9, 10) aminoacylated polynucleotides with several types of amino acid derivatives and obtained mixed results, some derivatives providing enrichment of the L-enantiomers, some of the D-, and others giving no enrichment at all (see below). To test the possibility of RNA-directed stereoselectivity further, space-filling models of RNA molecular interactions with D- and L--amino acids were constructed under various configurational restraints. It was readily apparent that RNA molecules in free solution, with no rotational constraints, should display little or no stereoselectivity for either D- or L-enantiomers of the amino acids. For RNA to have been uniquely stereoselective for the L-enantiomers, some other factors must have been involved.

It has been noted that the early RNA molecules would have been relatively unstable in fully aqueous environments. This has led to proposals that life evolved in organic surface layers instead of in bulk solution, thus providing an environment of lower water activity (11, 12). The successful abiotic polymerization of ribonucleotides on mineral surfaces has been described recently (13). To mimic these proposed surface conditions, the RNA/amino acid models were constructed on flat surfaces under constraints that maximized the degree of contact of the polar groups with the surface. This resulted in the bulky purine/pyrimidine bases and R group side chains of the amino acids becoming aligned above the plane of the surface, as illustrated in Fig. 1.

View larger version (29K): [in this window] [in a new window]   Figure 1. Surface configuration of aminoacylated D-ribose RNA. A dinucleotide segment of the RNA molecule is depicted as immobilized on a flat surface and viewed directly from above. The bulky purine/pyrimidine bases and the R group of the amino acid are above the plane of the surface. Due to steric interference with the -amino group and the constraint that the R group projects vertically, the aminoacylation reaction at the 2' hydroxyl is selective for the L-enantiomer of the amino acid, as depicted. In this configuration, the ribose rings are collinear; 80% of the polar oxygen groups in the structure are in contact with or oriented toward the surface. The majority of the nonpolar groups are oriented upward, above the plane.

Because both the 3' and 5' hydroxyl groups of RNA are involved in the sugar-phosphate backbone of the molecule, only the 2' hydroxyl group is available for aminoacylation. In the surface configuration depicted in Fig. 1and Fig. 2, it was found that steric interference with the -amino group prevents access of the D-isomers, so that incorporation of an amino acid by the 2' aminoacylation reaction would be selective for the L-enantiomers. Thus, if the RNA and the amino acids were constrained in surface monolayers, oriented with the nonpolar groups above the surface, incorporation of amino acid L-isomers would have automatically been favored.

View larger version (109K): [in this window] [in a new window]   Figure 2. Models of surface-bound D-ribose-L-aminoacyl-RNA. Models of the interaction between RNA and amino acids were constrained on a flat glass surface and photographed from above. The R group of the amino acid and the purine/pyrimidine bases are represented by small spheres so as not to obscure the underlying aminoacyl group. Because of the constraint that the R group remain above the plane of the surface, steric hindrance with the -amino group will restrict access of the D-isomers of the amino acids. In this surface configuration, therefore, aminoacylation of the 2'-hydroxyl group of RNA will automatically be selective for the L-enantiomers.

The stereochemical principle underlying this selectivity dictates that when reaction between an asymmetric entity (in this case, RNA) and a racemic mixture (in this case, DL-amino acids) is constrained to take place in only part (180°) of the possible 360° rotational space, then the reaction will automatically favor one stereoisomer over the other. This topological selectivity is reminiscent of the well-known `Ogston effect', in which symmetrical intermediates of the tricarboxylic acid cycle (fumarate and cis-aconitate) and pyruvate yield the asymmetric products L-malate, L-isocitrate, and L-lactate, but no D-isomers (14). In a similar fashion, models of L-RNA with D-and L-amino acids ( Fig. 3) showed that L-ribose RNA would have been stereoselective for D-amino acids.

View larger version (22K): [in this window] [in a new window]   Figure 3. Generation of D- vs. L-amino acid homochirality by surface-bound L-ribose vs. D-ribose RNA. The conditions are as described in Fig. 1. Note that surface-bound L-RNA would be stereoselective for D-enantiomers of the amino acids.

The current selection of amino acids for protein synthesis uses the 3' aminoacylated tRNA derivatives exclusively. However, for the estimated minimum size of a self-acylating RNA molecule (15), at least 98% of the free hydroxyl groups will have the 2' internal, or endo, configuration. The relative stereoselectivities of the 2' endo- and 2' and 3' exo-aminoacylations were therefore examined further. Models of the interactions of D- and L-amino acids with the 2' and 3' hydroxyl groups of surface-bound RNA were constructed ( Fig. 4 and Fig. 5). It was found that both the 2' endo-and 3' exo-aminoacylations of surface-bound RNA would be stereoselective for the L-enantiomers, but that the selectivity of only the endo- or internal aminoacylations would be absolute ( Fig. 4).

View larger version (24K): [in this window] [in a new window]   Figure 4. Positional stereoselectivity of RNA aminoacylation. The terminal dinucleotide segments of surface-bound D-ribose RNA are illustrated. The purine/pyrimidine bases (large open circles) and the R groups of the amino acids (shaded circles) are above the plane of the surface. In this conformation, only the 2' endo aminoacylation is completely stereoselective for the L-enantiomers.

View larger version (107K): [in this window] [in a new window]   Figure 5. Interaction of a surface-bound, single-stranded RNA segment with D- and L-amino acids. The 3' terminal tetranucleotide segment of a surface-bound, single-stranded RNA molecule is depicted, aminoacylated at a 2' endo position. The purine/pyrimidine bases are represented by small spheres so as not to obscure the underlying 2' hydroxyl groups. Note that a D-amino acid (upper right) will be unable to undergo 2' aminoacylation because of steric interference by the -amino group, whereas the L-amino acid below it has ready access to the 2' hydroxyl group.

Profy and Usher (9, 10) noted, during aminoacylation of oligonucleotides in bulk solution, that if enantiomeric enrichments occurred they were restricted to the 2' endo-hydroxyl groups. When the aminoacylations were carried out without an amino protective group, incorporation at the 2' position favored the D-enantiomers. When the 2, 3 dinitrobenzoyl group was used to protect the amino nitrogen, excesses of up to 60% of the L-enantiomer were obtained. It is possible that steric interactions of the bulky dinitrobenzoyl group with nitrogenous bases may have been involved in the reversal of selectivity, since enantiomeric excesses decreased from about 60% for polypurines to only 35% for polypyrimidine nucleotides; when the smaller N carboxy-anhydrides were used, no stereoselectivity was observed. Profy and Usher (10) concluded that stereochemical variability argued against a direct involvement in the early evolution of chirality, since either D- and L-amino acids could be selected and the presence of the N protective group precluded participation in a recursive mechanism for peptide bond formation.

Much of the RNA in a protobiotic world would probably be in the double-stranded form. However, because of increased steric interference introduced by a bulky group at the 2' position, the 2' aminoacylation of double-stranded RNA would be much more difficult than that of the single-stranded form. By forcing the ribose rings at an angle, the double-stranded helical configuration causes interference with the 2' hydroxyl group of the neighboring nucleotide. This steric interaction initially caused problems in adapting the Watson-Crick DNA model to RNA and would almost certainly hinder access of a bulky aminoacyl group. However, if aminoacylation of the double-stranded form of RNA were to occur, it might provide an asymmetry that would also favor L-enantiomers, particularly for amino acids having relatively large R groups (unpublished results). In the system proposed in this paper, the stereoselective asymmetry is introduced by the surface binding of single-stranded RNA. Adsorbing RNA on a polar or mineral surface relieves steric hindrance at the 2' position by allowing the ribose rings to become collinear and parallel to one another, and consequently less constrained than in the double-stranded form, as shown in Fig. 5. The ring oxygen and the 2' and 3' oxygens are coplanar and in contact with the surface, providing relatively easy access for the L-aminoacyl moiety at the 2' endo-hydroxyl group.

The selection of aminoacylated tRNAs for protein synthesis is determined by formation of the EF·Tu complex and favors the 3' aminoacylated species exclusively. The ribosomal peptidyl transferase is also completely specific for the 3' derivatives (16). However, as noted above, only the 2' endo-aminoacylation of RNA would have been stereoselective for L-amino acids. The current positional selectivity of tRNA synthetases for different amino acids was therefore surveyed in order to determine whether there was evidence of a positional change in aminoacylation of the D-ribose moiety. It was reported by Sprintzl and Cramer in 1970 (see ref. 17) that some of the synthetases catalyzed a primary 2' aminoacylation of tRNA (17). It has since been found that the 2' and 3' aminoacyl tRNA synthetases represent two distinct families of enzymes that differ fundamentally in their mechanism of action and the stereochemistry of their interaction with the RNA molecule. The class I enzymes approach and bind to the acceptor stem of tRNA from the minor groove side, resulting in a 2' aminoacylation, whereas the class II synthetases approach from the major groove side, producing the 3' aminoacylated species (17). As summarized in Table 1, a majority of present-day tRNAs still undergo a primary 2' aminoacylation. They are subsequently converted to the 3' derivatives by 2' 3' aminoacyl transferases before incorporation into protein.

The origin of this additional 2' aminoacylation step has never been explained. However, Moras (18) points out that the very different action patterns and family interrelationships of class I and class II synthetases indicate that a separation of the two groups arose quite early in evolution. The simple monomeric nature of class I synthetases contrasts with the complex multimeric structure of the class II enzymes (19, 20). Furthermore, the necessity for the 2' 3' acyl transferases also suggests that 2' aminoacylation may have arisen first. A discerned pattern in the types of amino acids served by the two groups of tRNA synthetases is consistent with this. For example, the smaller and more polar amino acids use the 3' position, which (as illustrated in Fig. 5) is oriented with the polar face of the RNA backbone, whereas aromatic amino acids and those with the largest and most nonpolar side chains continue to use the 2' position on the nonpolar face of the molecule as the primary site of aminoacylation. The existence of this, apparently redundant, initial 2' aminoacylation for most tRNAs, plus the necessity for the 2' 3' acyl transferases, could thus be explained if RNA aminoacylation first arose as a generalized 2' process, stereoselective for L-amino acids as outlined here. At some time after the establishment of homochirality, 3' aminoacylation then partially replaced the 2' process because of an evolved requirement for 3' aminoacylated tRNA (16).

The conclusions of this paper, which suggest a surface requirement for the origin of homochirality, reinforce previous theoretical studies (12) indicating that life may have originated in surface monolayers. In addition, since ribose is the only chiral center in RNA, L-amino acid homochirality was also predetermined by the selection of D-ribose, and not L-ribose, in the earliest RNA molecules. These conclusions have some interesting implications. First, the hypothesis should be testable, since it may be possible to duplicate the asymmetric abiotic aminoacylation of RNA under laboratory conditions. This could be done in several ways, perhaps by using the high-affinity binding of RNA to minerals such as hydroxyapatite in combination with either chemical condensation (using carbodiimides or cyanamide) or the auto-aminoacylating reaction with aminoacyl AMP described by Illangasekare et al. (15) Second, if the development of primitive life elsewhere was a fairly common event and had followed a path similar to that on Earth, evolution of L-ribose RNA would have led to D-amino acid homochirality. Consequently, if the selection of D-ribose rather than L-ribose was a chance event, then the presence of enantiomeric excesses of either D- or L-amino acids in different extraterrestrial biogenic sources [meteorites (21), comets, Martian samples (22), etc.] would be possible.

	FOOTNOTES

 
¹ Correspondence: Department of Biochemistry and Molecular Biology, The George Washington University, 2300 Eye St., N.W., Washington, DC 20037, USA.

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