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.     

Assembly of a class I tRNA synthetase from products of an artificially split gene

The aminoacyl-tRNA synthetases arose early in evolution and established the rules of the genetic code through their specific interactions with amino acids and RNA molecules. About half of these tRNA charging enzymes are class I synthetases, which contain similar N-terminal nucleotide-fold-like structures that are joined to variable domains implicated in specific protein-tRNA contacts. Here, we show that a bacterial synthetase gene can be split into two nonoverlapping segments. We split the gene for Escherichia coli methionyl-tRNA synthetase (a class I synthetase) at several sites near the interdomain junction, such that one segment codes for the nucleotide-fold-containing domain and the other provides determinants for tRNA recognition. When the segments are folded together, they can recognize and charge tRNA, both in vivo and in vitro. We postulate that an early step in the assembly of systems to attach amino acids to specific RNA molecules may have involved specific interactions between discrete proteins that is reflected in the interdomain contacts of modern synthetases.     

On the dimerization of the primitive tRNAs: implications in the origin of genetic code

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.     

Transfer RNA recognition by aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases are an ancient group of enzymes that catalyze the covalent attachment of an amino acid to its cognate transfer RNA. The question of specificity, that is, how each synthetase selects the correct individual or isoacceptor set of tRNAs for each amino acid, has been referred to as the second genetic code. A wealth of structural, biochemical, and genetic data on this subject has accumulated over the past 40 years. Although there are now crystal structures of sixteen of the twenty synthetases from various species, there are only a few high resolution structures of synthetases complexed with cognate tRNAs. Here we review briefly the structural information available for synthetases, and focus on the structural features of tRNA that may be used for recognition. Finally, we explore in detail the insights into specific recognition gained from classical and atomic group mutagenesis experiments performed with tRNAs, tRNA fragments, and small RNAs mimicking portions of tRNAs.     

On the Evolution of Structure in Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

On the evolution of structure in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

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.     

Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.     

Functional expansion of human tRNA synthetases achieved by structural inventions

Known as an essential component of the translational apparatus, the aminoacyl-tRNA synthetase family catalyzes the first step reaction in protein synthesis, that is, to specifically attach each amino acid to its cognate tRNA. While preserving this essential role, tRNA synthetases developed other roles during evolution. Human tRNA synthetases, in particular, have diverse functions in different pathways involving angiogenesis, inflammation and apoptosis. The functional diversity is further illustrated in the association with various diseases through genetic mutations that do not affect aminoacylation or protein synthesis. Here we review the accumulated knowledge on how human tRNA synthetases used structural inventions to achieve functional expansions.     

Evolution of the aminoacyl-tRNA synthetases and the origin of the genetic code

The aminoacyl-tRNA synthetases exist as two enzyme families which were apparently generated by divergent evolution from two primordial synthetases. The two classes of enzymes exhibit intriguing familial relationships, in that they are distributed nonrandomly within the codon-amino acid matrix of the genetic code. For example, all XCX codons code for amino acids handled by class II synthetases, and all but one of the XUX codons code for amino acids handled by class I synthetases. One interpretation of these patterns is that the synthetases coevolved with the genetic code. The more likely explanation, however, is that the synthetases evolved in the context of an already-established genetic code—a code which developed earlier in an RNA world. The rules which governed the development of the genetic code, and led to certain patterns in the coding catalog between codons and amino acids, would also have governed the subsequent evolution of the synthetases in the context of a fixed code, leading to patterns in synthetase distribution such as those observed. These rules are (1) conservative evolution of amino acid and adapter binding sites and (2) minimization of the disruptive effects on protein structure caused by codon meaning changes.     

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is ‘orthogonal’, meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA^Glu sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.     

The specificity of aminoacylation: a tRNA-tRNA interaction model.

Most of the isoacceptor species for a particular tRNA can be classified according to the middle base in the anticodon together with the fourth base in the amino acid stem. These specifying nucleotides would operate if a tRNA-tRNA interaction occurs on the aminoacyl-tRNA synthetase so that the anticodon of one tRNA molecule faces the fourth base of the other tRNA molecule. This model explains most of the misacylation reactions or changes in aminoacylation after mutation or chemical modifications of tRNAs. It also provides an explanation for biochemical properties of the aminoacyl-tRNA synthetases such as the presence of two active sites, and for the high fidelity of the aminoacylation. It may give insight into the origin and stability of the genetic code.     

Professor Tatsuo Miyazawa: from molecular structure to biological function

The late Prof. Tatsuo Miyazawa was an outstanding physical chemist, who established a number of spectroscopic methods to analyse the structures of proteins, peptides and nucleotides, and used them to understand molecular functions. He developed an infrared spectroscopic method to quantitatively analyse the secondary structures, α-helices and β-strands, of proteins. He successfully utilized nuclear magnetic resonance (NMR) methods to determine the conformations of peptides and proteins, particularly with respect to the interactions with their target molecules, which served as a solid basis for the wide range of applications of NMR spectroscopy to life science research. For example, he found that physiologically active peptides are randomly flexible in solution, but assume a particular effective conformation upon binding to their functional environments, such as membranes. He also used NMR spectroscopy to quantitatively analyse the conformer equilibrium of nucleotides, and related the dynamic properties of the modified nucleosides naturally-occurring in transfer ribonucleic acids (tRNAs) to their roles in correct codon recognition in protein synthesis. Furthermore, he studied the mechanisms of protein biosynthesis systems, including tRNA and aminoacyl-tRNA synthetases. Inspired by the structural mechanism of amino acid recognition by aminoacyl-tRNA synthetases, as revealed by NMR spectroscopy, he initiated a new research area in which non-natural amino acids are site-specifically incorporated into proteins to achieve novel protein functions (alloprotein technology).     

Editing of errors in selection of amino acids for protein synthesis.

All living cells must conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from gene to finished protein product. One crucial "quality control" point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. During selection of amino acids, synthetases very often have to distinguish the cognate substrate from a homolog having just one fewer methyl group in its structure. The binding energy of a methyl group is estimated to contribute only a factor of 100 to the specificity of binding, yet synthetases distinguish such closely related amino acids with a discrimination factor of 10,000 to 100,000. Examples of this include methionine versus homocysteine, isoleucine versus valine, alanine versus glycine, and threonine versus serine. Many investigators have demonstrated in vitro the ability of certain aminoacyl-tRNA synthetases to edit, that is, correct or prevent incorrect attachment of amino acids to tRNA molecules. Several major editing pathways are now established from in vitro data. Further, at least some aminoacyl-tRNA synthetases have recently been shown to carry out the editing function in vivo. Editing has been demonstrated to occur in both Escherichia coli and Saccharomyces cerevisiae. Significant energy is expended by the cell for editing of misactivated amino acids, which can be reflected in the growth rate. Because of this, cellular levels of aminoacyl-tRNA synthetases, as well as amino acid biosynthetic pathways which yield competing substrates for protein synthesis, must be carefully regulated to prevent excessive editing. High-level expression of recombinant proteins imposes a strain on the biosynthetic capacity of the cell which frequently results in misincorporation of abnormal or wrong amino acids owing in part to limited editing by synthetases. Unbalanced amino acid pools associated with some genetic disorders in humans may also lead to errors in tRNA aminoacylation. The availability of X-ray crystallographic structures of some synthetases, combined with site-directed mutagenesis, allows insights into molecular details of the extraordinary selectivity of synthetases, including the editing function.     

Genomics and the evolution of aminoacyl-tRNA synthesis.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.     

On the Classes of Aminoacyl-tRNA Synthetases,Amino Acids and the Genetic Code

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering models for the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporation of new amino acids following duplication and divergence of previous synthetases and tRNA molecules.     

Quality control in tRNA charging -- editing of homocysteine

All living organisms conduct protein synthesis with a high degree of accuracy maintained in the transmission and flow of information from a gene to protein product. One crucial 'quality control' point in maintaining a high level of accuracy is the selectivity by which aminoacyl-tRNA synthetases furnish correctly activated amino acids, attached to tRNA species, as the building blocks for growing protein chains. When differences in binding energies of amino acids to an aminoacyl-tRNA synthetase are inadequate, editing is used as a major determinant of enzyme selectivity. Some incorrect amino acids are edited at the active site before the transfer to tRNA (pre-transfer editing), while others are edited after transfer to tRNA at a separate editing site (post-transfer editing). Access of natural non-protein amino acids, such as homocysteine, homoserine, or ornithine to the genetic code is prevented by the editing function of aminoacyl-tRNA synthetases. Disabling editing function leads to tRNA mischarging errors and incorporation of incorrect amino acids into protein, which is detrimental to cell homeostasis and inhibits growth. Continuous homocysteine editing by methionyl-tRNA synthetase, resulting in the synthesis of homocysteine thiolactone, is part of the process of tRNA aminoacylation in living organisms, from bacteria to man. Excessive homocysteine thiolactone synthesis in hyperhomocysteinemia caused by genetic or nutritional deficiencies is linked to human vascular and neurological diseases.     

Basic faced alpha-helices are widespread in the peptide extensions of the eukaryotic aminoacyl-tRNA synthetases

Three aminoacyl-tRNA synthetases from yeast, one from plants and one from mammals possess unusual structures at their N termini, namely alpha helices with basic residues distributed asymmetrically, on a single face of the helix. It is unknown if these 'basic faced' alpha helices (BFAHs) are unique to the aminoacyl-tRNA synthetases. Analysis of the amino acid sequences of these five aminoacyl-tRNA synthetases using the hydrophobic moment algorithm failed to accurately identify the BFAHs. A new algorithm was therefore developed, called the 'basic moment'. This is a Fourier analysis procedure that predicts the distribution of basic residues within protein secondary structure. The basic moment identifies with a high degree of accuracy the five known BFAHs and also identifies further potential BFAHs at evolutionarily conserved positions in the peptide extensions of aspartyl-, lysyl- and valyl- tRNA synthetases from a range of eukaryotic species. In addition, the algorithm identifies the two-helix pair tRNA binding domain of alanyl-tRNA synthetase, implying that the domain includes a BFAH. The functional and evolutionary aspects of these structural features are discussed.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

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.