On the origin of the genetic code: signatures of its primordial complementarity in tRNAs and aminoacyl-tRNA synthetases

If the table of the genetic code is rearranged to put complementary codons face-to-face, it becomes apparent that the code displays latent mirror symmetry with respect to two sterically different modes of tRNA recognition. These modes involve distinct classes of aminoacyl-tRNA synthetases (aaRSs I and II) with recognition from the minor or major groove sides of the acceptor stem, respectively. We analyze the anticodon pairs complementary to the face-to-face codon couplets. Taking into account the invariant nucleotides on either side (5' and 3'), we consider the risk of anticodon confusion and subsequent erroneous aminoacylation in the ancestral coding system. This logic leads to the conclusion that ribozymic precursors of tRNA synthetases had the same two complementary modes of tRNA aminoacylation. This surprising case of molecular mimicry (1) shows a key potential selective advantage arising from the partitioning of aaRSs into two classes, (2) is consistent with the hypothesis that the two aaRS classes were originally encoded by the complementary strands of the same primordial gene and (3) provides a 'missing link' between the classic genetic code, embodied in the anticodon, and the second, or RNA operational, code that is embodied mostly in the acceptor stem and is directly responsible for proper tRNA aminoacylation.     

Revisiting the operational RNA code for amino acids: Ensemble attributes and their implications

It has been suggested that tRNA acceptor stems specify an operational RNA code for amino acids. In the last 20 years several attributes of the putative code have been elucidated for a small number of model organisms. To gain insight about the ensemble attributes of the code, we analyzed 4925 tRNA sequences from 102 bacterial and 21 archaeal species. Here, we used a classification and regression tree (CART) methodology, and we found that the degrees of degeneracy or specificity of the RNA codes in both Archaea and Bacteria differ from those of the genetic code. We found instances of taxon-specific alternative codes, i.e., identical acceptor stem determinants encrypting different amino acids in different species, as well as instances of ambiguity, i.e., identical acceptor stem determinants encrypting two or more amino acids in the same species. When partitioning the data by class of synthetase, the degree of code ambiguity was significantly reduced. In cryptographic terms, a plausible interpretation of this result is that the class distinction in synthetases is an essential part of the decryption rules for resolving the subset of RNA code ambiguities enciphered by identical acceptor stem determinants of tRNAs acylated by enzymes belonging to the two classes. In evolutionary terms, our findings lend support to the notion that in the pre-DNA world, interactions between tRNA acceptor stems and synthetases formed the basis for the distinction between the two classes; hence, ambiguities in the ancient RNA code were pivotal for the fixation of these enzymes in the genomes of ancestral prokaryotes.     

One ancestor for two codes viewed from the perspective of two complementary modes of tRNA aminoacylation

Background  

The genetic code is brought into action by 20 aminoacyl-tRNA synthetases. These enzymes are evenly divided into two classes (I and II) that recognize tRNAs from the minor and major groove sides of the acceptor stem, respectively. We have reported recently that: (1) ribozymic precursors of the synthetases seem to have used the same two sterically mirror modes of tRNA recognition, (2) having these two modes might have helped in preventing erroneous aminoacylation of ancestral tRNAs with complementary anticodons, yet (3) the risk of confusion for the presumably earliest pairs of complementarily encoded amino acids had little to do with anticodons. Accordingly, in this communication we focus on the acceptor stem.     

Aminoacylation of RNA Minihelices: Implications for tRNA Synthetase Structural Design and Evolution

Abstract

The genetic code is based on the aminoacylation of tRNA with amino acids catalyzed by the aminoacyl-tRNA synthetases. The synthetases are constructed from discrete domains and all synthetases possess a core catalytic domain that catalyzes amino acid activation, binds the acceptor stem of tRNA, and transfers the amino acid to tRNA. Fused to the core domain are additional domains that mediate RNA interactions distal to the acceptor stem. Several synthetases catalyze the aminoacylation of RNA oligonucleotide substrates that recreate only the tRNA acceptor stems. In one case, a relatively small catalytic domain catalyzes the aminoacylation of these substrates independent of the rest of the protein. Thus, the active site domain may represent a primordial synthetase in which polypeptide insertions that mediate RNA acceptor stem interactions are tightly integrated with determinants for aminoacyl adenylate synthesis. The relationship between nucleotide sequences in small RNA oligonucleotides and the specific amino acids that are attached to these oligonucleotides could constitute a second genetic code.     

Genetic perturbations of RNA reveal structure-based recognition in protein-RNA interaction

Protein-RNA recognition is an essential foundation of cellular processes, yet much remains unknown about these important interactions. The recognition between aminoacyl-tRNA synthetases and their cognate tRNA substrates is highly specific and essential for cell viability, due to the necessity for accurate translation of the genetic code into protein sequences. We selected an active tRNA that is highly mutated in the recognition nucleotides of the acceptor stem region in the alanine system. The functional properties of this mutant and its secondary derivatives demonstrate that recognition cannot be reduced to isolated structural elements, but rather the amino acid acceptor stem is being recognized as a unit.     

Translation of both complementary strands might govern early evolution of the genetic code

The updated structural and phylogenetic analyses of tRNA pairs with complementary anticodons provide independent support for our earlier finding, namely that these tRNA pairs concertedly show complementary second bases in the acceptor stem. Two implications immediately follow: first, that a tRNA molecule gained its present, complete, cloverleaf shape via duplication(s) of a shorter precursor. Second, that common ancestry is shared by two major components of the genetic code within the tRNA molecule--the classic code per se embodied in anticodon triplets, and the operational code of aminoacylation embodied primarily in the first three base pairs of the acceptor stems. In this communication we show that it might have been a double, sense-antisense, in-frame translation of the very first protein-encoding genes that directed the code's earliest expansion, thus preserving this fundamental dual-complementary link between acceptors and anticodons. Furthermore, the dual complementarity appears to be consistent with two mirror-symmetrical modes by which class I and II aminoacyl-tRNA synthetases recognize the cognate tRNAs--from the minor and major groove side of the acceptor stem, respectively.     

Origin of the genetic code: first aminoacyl-tRNA synthetases could replace isofunctional ribozymes when only the second base of codons was established

Analysis of the updated compilation of more than 8,000 tRNA gene sequences confirmed our previously reported finding that in pairs of consensus tRNAs with complementary anticodons, their second bases in the acceptor stems are also complementary. This dual complementarity points to the following: (1) the operational code embodied in the acceptor stem, and the classic genetic code embodied in the anticodon could have had the same common ancestor; (2) new tRNAs most likely entered primitive translation in pairs with complementary anticodons; and (3) this process of code expansion was directed by the primordial double-strand coding. However, we did not find the dual complementarity when testing all tRNA pairs in which anticodons were complementary only at the central position, but not complementary at least at one of the flanking two positions. This observation, together with certain additional evidence, suggests that both codes were still being shaped (with only the second base established at the time) when the first protein aminoacyl-tRNA synthetases could have already started replacing their ribozymic precursors.     

Small RNA helices as substrates for aminoacylation and their relationship to charging of transfer RNAs.

RNA microhelices that reconstruct the acceptor stems of transfer RNAs can be aminoacylated. The anticodon-independent aminoacylation is sequence-specific and suggests a relationship between amino acids and nucleotide sequences which is different from that of the classical genetic code. The specific aminoacylation of RNA microhelices also suggests a highly differentiated adaptation of the structures of aminoacyl-tRNA synthetases to sequences in the acceptor stems of transfer RNAs.     

Statistical evidence for remnants of the primordial code in the acceptor stem of prokaryotic transfer RNA

Summary The specificity of interaction of amino acids with triplets in the acceptor helix stem of tRNA was investigated by means of a statistical analysis of 1400 tRNA sequences. The imprint of a prototypic genetic code at position 3–5 of the acceptor helix was detected, but only for those major amino acids, glycine, alanine, aspartic acid, and valine, that are formed by spark discharges of simple gases in the laboratory. Although remnants of the code at position 3–5 are typical for tRNAs of archaebacteria, eubacteria, and chloroplasts, eukaryotes do not seem to contain this code, and mitochondria take up an intermediary position. A duplication mechanism for the transposition of the original 3–5 code toward its present position in the anticodon stern of tRNA is proposed. From this viewpoint, the mode of evolution of mRNA and functional ribosomes becomes more understandable.Offprint requests to: W. Moller     

Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases.

The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity.     

Structure of the acceptor stem of Escherichia coli tRNA Ala: role of the G3.U70 base pair in synthetase recognition.

The fidelity of translation of the genetic code depends on accurate tRNA aminoacylation by cognate aminoacyl-tRNA synthetases. Thus, each tRNA has specificity not only for codon recognition, but also for amino acid identity; this aminoacylation specificity is referred to as tRNA identity. The primary determinant of the acceptor identity of Escherichia coli tRNAAlais a wobble G3.U70 pair within the acceptor stem. Despite extensive biochemical and genetic data, the mechanism by which the G3.U70 pair marks the acceptor end of tRNAAla for aminoacylation with alanine has not been clarified at the molecular level. The solution structure of a microhelix derived from the tRNAAla acceptor end has been determined at high precision using a very extensive set of experimental constraints (approximately 32 per nt) obtained by heteronuclear multidimensional NMR methods. The tRNAAla acceptor end is overall similar to A-form RNA, but important differences are observed. The G3.U70 wobble pair distorts the conformation of the phosphodiester backbone and presents the functional groups of U70 in an unusual spatial location. The discriminator base A73 has extensive stacking overlap with G1 within the G1.C72 base pair at the end of the double helical stem and the -CCA end is significantly less ordered than the rest of the molecule.     

On the 28-gon symmetry inherent in the genetic code intertwined with aminoacyl-tRNA synthetases—The Lucas series

Despite considerable efforts it has remained unclear what principle governs the selection of the 20 canonical amino acids in the genetic code. Based on a previous study of the 28-gonal and rotational symmetric arrangement of the 20 amino acids in the genetic code, new analyses of the organization of the genetic code system together with their intrinsic relation to the two classes of aminoacyl-tRNA synthetases are reported in this work. A close inspection revealed how the enzymes and the 20 gene-encoded amino acids are intertwined on the polyhedron model. Complementary and cooperative symmetries between class I and class II aminoacyl-tRNA synthetases displayed by a 28-gon organization are discussed, and we found that the two previously suggested evolutionary axes within the genetic code overlap the symmetry axes within the two classes of aminoacyl-tRNA synthetases. Moreover, it has been shown that the side-chain carbon-atom numbers (2, 1, 3, 4 and 7) in the overwhelming majority of the amino acids recognized by each of the two classes of aminoacyl-tRNA synthetases are determined by a mathematical relationship, the Lucas series. A stepwise co-evolutionary selection logic of the amino acids is manifested by the amino acid side-chain carbon-atom number balance at ‘17’, when grouping the genetic code doublets in the 28-gon organization. The number ‘17’ equals the sum of the initial five numbers in the Lucas series, which are 2, 1, 3, 4 and 7.     

Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase

Protein-RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure-function relationships involved in the interaction of the Escherichia coli tRNA(Asp) acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA(Asp) complex. Mutants were derived from Saccharomyces cerevisiae tRNA(Asp), which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA(Asp) knockout strain of E. coli. The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA(Asp) gene. The mutants provide a rich source of tRNA(Asp) sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.     

The Seryl-tRNA synthetase/tRNA acceptor stem interface is mediated via a specific network of water molecules

tRNAs are aminoacylated by the aminoacyl-tRNA synthetases. There are at least 20 natural amino acids, but due to the redundancy of the genetic code, 64 codons on the mRNA. Therefore, there exist tRNA isoacceptors that are aminoacylated with the same amino acid, but differ in their sequence and in the anticodon. tRNA identity elements, which are sequence or structure motifs, assure the amino acid specificity. The Seryl-tRNA synthetase is an enzyme that depends on rather few and simple identity elements in tRNA^Ser. The Seryl-tRNA-synthetase interacts with the tRNA^Ser acceptor stem, which makes this part of the tRNA a valuable structural element for investigating motifs of the protein–RNA complex. We solved the high resolution crystal structures of two tRNA^Ser acceptor stem microhelices and investigated their interaction with the Seryl-tRNA-synthetase by superposition experiments. The results presented here show that the amino acid side chains Ser151 and Ser156 of the synthetase are interacting in a very similar way with the RNA backbone of the microhelix and that the involved water molecules have almost identical positions within the tRNA/synthetase interface.     

Translocation within the acceptor helix of a major tRNA identity determinant

The genetic code is defined by the specific aminoacylations of tRNAs by aminoacyl-tRNA synthetases. Although the synthetases are widely conserved through evolution, aminoacylation of a given tRNA is often system specific-a synthetase from one source will not acylate its cognate tRNA from another. This system specificity is due commonly to variations in the sequence of a critical tRNA identity element. In bacteria and the cytoplasm of eukaryotes, an acceptor stem G3:U70 base pair marks a tRNA for aminoacylation with alanine. In contrast, Drosophila melanogaster (Dm) mitochondrial (mt) tRNA(Ala) has a G2:U71 but not a G3:U70 pair. Here we show that this translocated G:U and the adjacent G3:C70 are major determinants for recognition by Dm mt alanyl-tRNA synthetase (AlaRS). Additionally, G:U at the 3:70 position serves as an anti-determinant for Dm mt AlaRS. Consequently, the mitochondrial enzyme cannot charge cytoplasmic tRNA(Ala). All insect mitochondrial AlaRSs appear to have split apart recognition of mitochondrial from cytoplasmic tRNA(Ala) by translocation of G:U. This split may be essential for preventing introduction of ambiguous states into the genetic code.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

The crystal structure of a Thermus thermophilus tRNA(Gly) acceptor stem microhelix at 1.6 Å resolution

tRNAs are aminoacylated with the correct amino acid by the cognate aminoacyl-tRNA synthetase. The tRNA/synthetase systems can be divided into two classes: class I and class II. Within class I, the tRNA identity elements that enable the specificity consist of complex sequence and structure motifs, whereas in class II the identity elements are assured by few and simple determinants, which are mostly located in the tRNA acceptor stem. The tRNA(Gly)/glycyl-tRNA-synthetase (GlyRS) system is a special case regarding evolutionary aspects. There exist two different types of GlyRS, namely an archaebacterial/human type and an eubacterial type, reflecting the evolutionary divergence within this system. We previously reported the crystal structures of an Escherichia coli and of a human tRNA(Gly) acceptor stem microhelix. Here we present the crystal structure of a thermophilic tRNA(Gly) aminoacyl stem from Thermus thermophilus at 1.6? resolution and provide insight into the RNA geometry and hydration.