Biochemical research on oogenesis. Aminoacyl tRNA turns over in the 42-S particles of Xenopus laevis oocytes, but its ester bond is protected against hydrolysis

The ester bond aminoacyl tRNA is protected against hydrolysis in the 42-S particles (thesaurisomes) present in Xenopus laevis previtellogenic oocytes. Deacylation of tRNA is very slow in vitro, unless ATP is present. ATP causes a partial turnover of aminoacyl tRNA in vitro, with no detectable decrease in the overall aminoacylation level of tRNA, which remains close to 100%. tRNA in the particles turns over rapidly in vivo. Since the ester bond of aminoacyl tRNA is stabilized inside the 42-S particles, this turnover cannot be a consequence of spontaneous deacylation of tRNA, followed by reacylation by the aminoacyl-tRNA synthetases associated with the particles. We rather consider this turnover as reflecting a true metabolic activity of the particles, and a direct or indirect involvement of these particles in the oocyte's protein-synthesizing system.     

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.     

Autoantibodies to aminoacyl-transfer RNA synthetases for isoleucine and glycine. Two additional synthetases are antigenic in myositis

Autoantibodies to three of the aminoacyl-transfer RNA (tRNA) synthetases have been reported (for histidine, threonine, and alanine). Most patients with these autoantibodies have polymyositis, and the majority also have interstitial lung disease. This study examined the question of whether autoantibodies to other aminoacyl-tRNA synthetases occur in the sera of myositis patients. We tested sera from patients with myositis with unidentified anticytoplasmic antibodies that immunoprecipitate tRNA for the ability to inhibit the aminoacyl-tRNA synthetases for the remaining 17 amino acids. Three sera showed strong inhibitory activity for a synthetase. OJ and NJ sera (and IgG) significantly inhibited isoleucyl-tRNA synthetase activity, each with 94% inhibition at the screening dilution, whereas other test sera and controls all inhibited less than 50%. OJ and NJ sera immunoprecipitated identical patterns of tRNA, and identical, complex patterns of high m.w. polypeptides that were consistent with the multienzyme synthetase complex of which isoleucyl-tRNA synthetase is a part. EJ serum (and IgG) significantly inhibited glycyl-tRNA synthetase, and immunoprecipitated a unique pattern of transfer RNA, and a strong predominant protein band of 77 kDa. These data strongly suggest that OJ and NJ have autoantibodies to isoleucyl-tRNA synthetase, and that EJ has antibodies to glycyl-tRNA synthetase. The findings of signs of muscle involvement in all three patients, and severe interstitial lung disease in OJ, strengthens the association of antisynthetases with these conditions.     

Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver.

A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.     

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.     

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.     

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.     

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.     

Active aminoacyl-tRNA synthetases are present in nuclei as a high molecular weight multienzyme complex

Recent studies suggest that aminoacylation of tRNA may play an important role in the transport of these molecules from the nucleus to the cytoplasm. However, there is almost no information regarding the status of active aminoacyl-tRNA synthetases within the nuclei of eukaryotic cells. Here we show that at least 13 active aminoacyl-tRNA synthetases are present in purified nuclei of both Chinese hamster ovary and rabbit kidney cells, although their steady-state levels represent only a small percentage of those found in the cytoplasm. Most interestingly, all the nuclear aminoacyl-tRNA synthetases examined can be isolated as part of a multienzyme complex that is more stable, and consequently larger, than the comparable complex isolated from the cytoplasm. These data directly demonstrate the presence of active aminoacyl-tRNA synthetases in mammalian cell nuclei. Moreover, their unexpected structural organization raises important questions about the functional significance of these multienzyme complexes and whether they might play a more direct role in nuclear to cytoplasmic transport of tRNAs.     

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.     

Amino acid selectivity in the aminoacylation of coenzyme A and RNA minihelices by aminoacyl-tRNA synthetases

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TpsiC helix of tRNA(Ile) or tRNA(Val) were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.     

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 exchange of the discriminator base A73 for G is alone sufficient to convert human tRNA(Leu) into a serine-acceptor in vitro.

Transfer RNA (tRNA) identify is maintained by the highly specific interaction of a few defined nucleotides or groups of nucleotides, called identity elements, with the cognate aminoacyl-tRNA synthetase, and by nonproductive interactions with the other 19 aminoacyl-tRNA synthetases. Most tRNAs have a set of identity elements in at least two locations, commonly in the anticodon loop or in the acceptor stem, and at the discriminator base position 73. We have used T7 RNA polymerase transcribed tRNAs to demonstrate that the sole replacement of the discriminator base A73 of human tRNA(Leu) with the tRNA(Ser)-specific G generates a complete identity switch to serine acceptance. The reverse experiment, the exchange of G73 in human tRNA(Ser) for the tRNA(Leu-specific A, causes a total loss of serine specificity without creating any leucine acceptance. These results suggest that the discriminator base A73 of human tRNA(Leu) alone protects this tRNA against serylation by seryl-tRNA synthetase. This is the first report of a complete identity switch caused by an exchange of the discriminator base alone.     

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.     

Effect of the overproduction of phenylalanyl- and threonyl-tRNA synthetases on tRNAPhe and tRNAThr concentrations in E. coli cells

Transformation of an E. coli strain with a recombinant plasmid DNA (pB1) encoding the genes for phenylalanyl- and threonyl-tRNA synthetases causes overproduction of these enzymes by about 100- and 5-fold, respectively. A possible effect of the overproduction of the two aminoacyl-tRNA synthetases on intracellular cognate tRNA levels has been searched for by comparing tRNAThr and tRNAPhe aminoacylation capacities in the RNA extracts from strains carrying pB1 or pBR322 plasmid DNA. The answer is that the levels of these tRNAs are not changed by selective increase of the cognate synthetases.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.     

Growth-dependent factors in the regulation of aminoacyl-tRNA synthetase activities of Tetrahymena pyriformis.

Changes in phenylalanyl-tRNA synthetase (L-phenylalanine : tRNAPhe ligase, EC 6.1.1.20) and leucyl-tRNA synthetase (L-leucine : tRNALeu ligase. EC 6.1.1.4) activities were studied during the growth cycle of Tetrahymena pyriformis. High levels of charged tRNA observed during exponential growth were associated with elevated aminoacyl-tRNA synthetase activities. Low levels of charges tRNA in the stationary phase culture were associated with decreased aminoacyl-tRNA synthethase activities together with a concomitant accumulation of factor(s) which inhibited the enzyme activities. The inhibitory factor(s) has been partially purified and evidence is presented to rule out RNA, RNAases, proteases and ATPases as the responsible inhibitory factor(s) of the aminoacyl-tRNA synthetases.