Isolation of transfer RNA isoacceptors by chromatography on dihydroxyboryl-substituted cellulose,polyacrylamide, and glass

Polyacrylamide and porous-glass supports containing the dihydroxyborylphenyl group can be prepared by a method similar to that used in the synthesis of N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethylcellulose. The reaction of aminoethylpolyacrylamide or amino-substituted glass with N-(m-dihydroxyborylphenyl)succinamic acid in the presence of N-cyclohexyl-N′-β-(4-methyl-morpholinium) ethylcarbodiimide yields products which, together with the cellulose derivative, are all capable of binding tRNA dissolved in buffers at pH 8.7. The demonstration that bound tRNA can be released with sorbitol solutions or with low pH buffers, together with studies on the binding of tRNA species that contain chemically modified 3′-terminals, indicate that the predominant binding mechanism consists of cyclic complex formation between the immobilized dihydroxyboryl groups and the 3′-terminal cis-diol groups of the tRNA molecules. Aminoacylated tRNA does not bind under the conditions necessary to bind tRNA and this permits the isolation of specific tRNA isoacceptors. The purification of tRNA^Phe and the partial purification of tRNA^Glu and tRNA^Trp are described.     

Isolation of Escherichia coli precursor tRNAs containing modified nucleoside Q.

Affinity chromatography based on the complex formation of the modified nucleoside Q with boronic acid has been applied to the isolation of specific tRNA precursors containing this modified nucleoside. When [32P]RNA isolated from an Escherichia coli strain containing a thermolabile ribonuclease P was chromatographed on dihydroxyboryl-substituted cellulose, the precursors for asparagine, aspartate, histidine, and tyrosine tRNA were specifically retained. All precursors were monomeric. The nucleotide sequences of four asparagine tRNA precursors were determined.     

Superposition of a tRNASer acceptor stem microhelix into the seryl-tRNA synthetase complex

Aminoacyl-tRNA synthetases catalyze the formation of aminoacyl-tRNAs. Seryl-tRNA synthetase is a class II synthetase, which depends on rather few and simple identity elements in tRNA(Ser) to determine the amino acid specificity. tRNA(Ser) acceptor stem microhelices can be aminoacylated with serine, which makes this part of the tRNA a valuable tool for investigating the structural motifs in a tRNA(Ser)-seryl-tRNA synthetase complex. A 1.8A-resolution tRNA(Ser) acceptor stem crystal structure was superimposed to a 2.9A-resolution crystal structure of a tRNA(Ser)-seryl-tRNA synthetase complex for a visualization of the binding environment of the tRNA(Ser) microhelix.     

ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.     

Aminoacyl-tRNA synthetase-induced cleavage of tRNA.

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.     

Reversed-phase boronate chromatography for the separation of O-methylribose nucleosides and aminoacyl-tRNAs

Boronate forms an anionic complex with the cis-2′,3′ hydroxyls of unsubstituted ribonucleosides and the 3′-terminal adenosine of unacylated tRNAs, but not with ribosesubstituted nucleosides such as 2′-O-methylnucleosides and aminoacyl-tRNAs. We have synthesized phenyl boronates with hydrophobic side chains of about 1-nm-long and coated inert 10-μm solid beads of polychlorotrifluoroethylene with this material. This matrix complexes easily with compounds containing free cis-hydroxyls, but not with their O-alkyl or O-acyl derivatives. This permits the separation of mammalian and bacterial amino-acyl-tRNAs from uncharged tRNAs and O-methyl nucleosides from ribose-unsubstituted nucleosides in one chromatographic step, as the substituted members of each group do not undergo boronate complex formation and are thus not as much retarded in passing through the column. Complex formation between ribofuranoses and the boronate matrix appears to be enhanced by the hydrophobic “tail” of the boronate compound, by the high ionic environment of the solvent, and by the hydrophobic nature of the inert support. This method of one-step purification of tRNAs on reversed-phase boronate columns has been tested for several tRNAs specific for amino acids of different hydrophobicity and ionic character. The results indicate that each tRNA tested can be purified with appreciable purity (70–95%) and high yield (80%). However, recovery of the queuine base containing aminoacyl-tRNAs is only about 6% of the applied material. Several other boronate matrices have also been synthesized using cellulose, agarose. Sepharose, or porous glass beads as the inert support with different lengths of the spacer arm. Cellulose with a 1-nm-long spacer arm is satisfactory not only for the separation of aminoacyl-tRNAs and O-methylribose nucleosides, but also for the separation as a group of tRNAs containing the base of Q, queuine. However, other inert supports are unsatisfactory because of a non-specific binding of the tRNAs.     

Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex

Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.     

Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid

An affinity electrophoretic method has been developed to study the state of terminal phosphorylation of RNAs and the presence of the hypermodified base Q in tRNA. It is based on the copolymerization of acryloylaminophenylboronic acid into standard polyacrylamide gels and the interaction of this derivative with free cis-diol groups present in the RNA. In the case of terminal phosphorylation, free ribose groups are present either as such, or may be introduced by enzymatic reactions specific for a particular phosphorylation pattern (e.g. using T4 RNA ligase or guanylyltransferase). Additionally, tRNA species containing the Q base may be resolved from Q-lacking tRNAs by boronate affinity electrophoresis. The introduction of a non-destructive, one-step electrophoretic procedure not only offers an alternative to classical analytical methods, but also provides a means of isolating such populations of RNAs for which other methods are unavailable or are less convenient.     

The free yeast aspartyl-tRNA synthetase differs from the tRNA(Asp)-complexed enzyme by structural changes in the catalytic site, hinge region, and anticodon-binding domain

Aminoacyl-tRNA synthetases catalyze the specific charging of amino acid residues on tRNAs. Accurate recognition of a tRNA by its synthetase is achieved through sequence and structural signalling. It has been shown that tRNAs undergo large conformational changes upon binding to enzymes, but little is known about the conformational rearrangements in tRNA-bound synthetases. To address this issue the crystal structure of the dimeric class II aspartyl-tRNA synthetase (AspRS) from yeast was solved in its free form and compared to that of the protein associated to the cognate tRNA(Asp). The use of an enzyme truncated in N terminus improved the crystal quality and allowed us to solve and refine the structure of free AspRS at 2.3 A resolution. For the first time, snapshots are available for the different macromolecular states belonging to the same tRNA aminoacylation system, comprising the free forms for tRNA and enzyme, and their complex. Overall, the synthetase is less affected by the association than the tRNA, although significant local changes occur. They concern a rotation of the anticodon binding domain and a movement in the hinge region which connects the anticodon binding and active-site domains in the AspRS subunit. The most dramatic differences are observed in two evolutionary conserved loops. Both are in the neighborhood of the catalytic site and are of importance for ligand binding. The combination of this structural analysis with mutagenesis and enzymology data points to a tRNA binding process that starts by a recognition event between the tRNA anticodon loop and the synthetase anticodon binding module.     

Mechanism of the difference in the binding affinity of E. coli tRNAGln to glutaminyl-tRNA synthetase caused by noninterface nucleotides in variable loop

Aminoacyl-tRNA synthetases (ARSs) distinguish their cognate tRNAs from many other kinds of tRNAs, despite the very similar tertiary structures of tRNAs. Many researchers have supported the view that this recognition is achieved by intermolecular interactions between tRNA and ARS. However, one of the aptamers of Escherichia coli glutamine specific tRNA, var-AGGU, has a higher affinity to ARS than the wild-type, although the sequence difference only lies in the variable loop located on the opposite side of the binding interface with ARS. To understand the reason for the difference in affinity, we did molecular dynamics simulations on tRNAs and their complexes with ARS. We calculated the enthalpic and entropic contributions to the binding free energy with the molecular mechanics-Poisson-Boltzmann/surface area method and found that the entropic difference plays an important role in the difference in binding free energies. During the molecular dynamics simulations, dynamic rearrangements of hydrogen bonds occurred in the tertiary core region of the wild-type tRNA, whereas they were not observed in the free var-AGGU simulation. Since the internal mobility was suppressed upon complex formation with ARS, the entropy loss in the wild-type was larger than that of the aptamer. We therefore concluded that the sequence difference in the variable loop caused the difference in the internal mobility of the tertiary core region tRNAs and led to the difference in the affinity to ARS through the entropy term.     

Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNA^Trp and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3′ end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an α-helix of the unique N-terminal domain and the anticodon loop by an α-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.     

Kinetic quality control of anticodon recognition by a eukaryotic aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are an ancient class of enzymes responsible for the matching of amino acids with anticodon sequences of tRNAs. Eukaryotic tRNA synthetases are often larger than their bacterial counterparts, and several mammalian enzymes use the additional domains to facilitate assembly into a multi-synthetase complex. Human cysteinyl-tRNA synthetase (CysRS) does not associate with the multi-synthetase complex, yet contains a eukaryotic-specific C-terminal extension that follows the tRNA anticodon-binding domain. Here we show by mutational and kinetic analysis that the C-terminal extension of human CysRS is used to selectively improve recognition and binding of the anticodon sequence, such that the specificity of anticodon recognition by human CysRS is higher than that of its bacterial counterparts. However, the improved anticodon recognition is achieved at the expense of a significantly slower rate in the aminoacylation reaction, suggesting a previously unrecognized kinetic quality control mechanism. This kinetic quality control reflects an evolutionary adaptation of some tRNA synthetases to improve the anticodon specificity of tRNA aminoacylation from bacteria to humans, possibly to accommodate concomitant changes in codon usage.     

Interaction of rabbit reticulocyte elongation factor 1 with guanosine-triphosphate and aminoacyl-transfer ribonucleic acid

Elongation Factor 1 (EF-1) from rabbit reticulocytes interacts with GTP to form a complex that is retained on a nitrocellulose filter. EF-1 also interacts with GDP; however, the concentration of GDP required for maximal complex formation is higher than the concentration of GTP required and the extent of binding is lower. Interaction of EF-1 with GTP in the presence of various aminoacyl-tRNAs from rabbit liver or E. coli results in a 50–75% decrease in the amount of GTP complex retained on a filter. No reduction in the amount of GTP complex retained is observed with deacylated tRNA or with N-acetylphenylalanyl-tRNA. EF-1 is inactivated by heating at 37 °C in the presence of GTP. Aminoacyl-tRNA protects EF-1 from the inactivation observed in the presence of GTP. These data indicate that an interaction of reticulocyte EF-1 with GTP and aminoacyl-tRNA occurs; however, attempts to demonstrate the formation of a stable ternary complex by chromatography on Sephadex G-150 were unsuccessful. Also, no difference is observed between the rate of binding of aminoacyl-tRNA to reticulocyte ribosomes obtained with EF-1 and the rate obtained with EF-1 that had been incubated previously with GTP and aminoacyltRNA.     

Structure of Escherichia coli Arginyl-tRNA Synthetase in Complex with tRNAArg: Pivotal Role of the D-loop

Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNA^Arg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure–function understanding in prokaryotic tRNA^Arg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNA^Arg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNA^Arg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.     

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.     

Active site of lysyl-tRNA synthetase: structural studies of the adenylation reaction

Aminoacyl-tRNA synthetases play a key role in protein biosynthesis by catalyzing the specific aminoacylation of tRNA. The energy required for the formation of the ester bond between the amino acid carboxylate group and the tRNA acceptor stem is supplied by coupling the reaction to the hydrolysis of ATP. Lysyl-tRNA synthetase from Escherichia coli belongs to the family of class II synthetases and carries out a two-step reaction, in which lysine is activated by being attached to the alpha-phosphate of AMP before being transferred to the cognate tRNA. Crystals of the thermo-inducible E. coli lysyl-tRNA synthetase LysU which diffract to 2.1 A resolution have been used to determine crystal structures of the enzyme in the presence of lysine, the lysyl-adenylate intermediate, and the nonhydrolyzable ATP analogue AMP-PCP. Additional data have been obtained from crystals soaked in a solution containing ATP and Mn(2+). The refined crystal structures give "snapshots" of the active site corresponding to key steps in the aminoacylation reaction and provide the structural framework for understanding the mechanism of lysine activation. The active site of LysU is shaped to position the substrates for the nucleophilic attack of the lysine carboxylate on the ATP alpha-phosphate. No residues are directly involved in catalysis, but a number of highly conserved amino acids and three metal ions coordinate the substrates and stabilize the pentavalent transition state. A loop close to the catalytic pocket, disordered in the lysine-bound structure, becomes ordered upon adenine binding.     

不同年龄小白鼠全脑细胞质氨酰tRNA合成酶活力的比较

从不同年龄(20天,30天,1年)的小白鼠全脑制得细胞质混合氨酰tRNA合成酶。用异源体系(即用酵母tRNA和小白鼠全脑氨酰tRNA合成酶)测定了氨酰tRNA合成酶分别载运~3H标记的Asp、Gly、Glu、Lys和Ala的活力。结果表明除未检出tRNA~(Glu)的合成酶活力外,对其余四种氨基酸都有明显的活力,特别是年龄20天小白鼠的氨酰tRNA合成酶对~3H-Gly具有高达35％的载运活力。对~3H-Gly、~3H-Lys和~3H-Ala的载运活力有随增龄而下降的趋势,但对~3H-Asp的载运活力则随年龄增长而增高。     

The presence of transfer RNA in the axoplasm of the squid giant axon

Previous work has revealed that 4S RNA is the primary species of RNA in the axoplasm from the giant axons of the squid and Myxicola. This study shows that axoplasmic 4S RNA from the squid giant axon has the functional properties of tRNA. Axoplasmic RNA was charged with amino acids by aminoacyl-tRNA synthetases prepared from squid brain. The aminoacylation was prevented by incubating the RNA with RNase prior to running the reaction. The amino acid-RNA complex was labile at pH 9, which is characteristic of the acyl linkage between an amino acid and its tRNA. Aminoacyl-tRNA synthetase activity was also present in the axoplasm, primarily in the soluble fraction.     

Chromatographic behavior of several mammalian tRNAs on acylated dihydroxyl-borate cellulose and Aminex A-28.

Studies of the chromatographic behavior of mammalian tRNAs, from several sources, on acylated DBAE-cellulose indicate that species of tRNA Asn , tRNA Asp and tRNA His can be retained on this matrix, while species of tRNA Tyr, tRNA Asn and tRNA Asp are not retained. Treatment of total rat liver tRNA with cyanogen bromide and subsequent chromatography on Aminex A-28 columns demonstrated that these tRNA species might contain Q (or Q*) nucleoside. However, comparable studies of the tRNA isolated from Walker 256 rat mammary tumor tissue demonstrated that this tumor tRNA almost totally lacks the hypermodified nucleosides Q and Q*. In addition, we have found that at least the major species of rat liver tRNA Asn contains the Q nucleoside. These studies indicate that chromatography on the acylated DBAE-cellulose matrix, couple with the analytical ion-exchange chromatography of cyanogen bromide treated and untreated amino-acyl-tRNA can be a valuable technique for the determination of alterations in the Q (or Q*) nucleoside content of the tRNAs isolated from normal and tumor tissues.     

tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.