Antico-operative binding of bacterial and mammalian initiator tRNAMet to methionyl-tRNA synthetase from Escherichia coli

The reaction scheme of methionyl-tRNA synthetase from Escherichia coli with the initiator tRNA_s^Met from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, k_R, and for the dissociation, k_D, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical k_R values of 250 μm^?1 s^?1 reflect similar behaviour for antico-operative binding of both tRNA_s^Met to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNA_s^Met to measure the mode of exchange of these tRNAs antico-operatively bound to the enzyme. Analysis of the results reveals that antico-operativity does not arise from structural asymmetric assembly of the enzyme subunits. Indeed, both subunits can potentially bind a tRNA molecule. Exchange between tRNA molecules can occur via a transient complex in which both sites are occupied. Either strong and weak sites reciprocate between subunits on the transient complex or occupation of the weak site induces symmetry of this complex. While in the present case, these two alternatives are kinetically indistinguishable, they do account for the observation that, upon increasing the concentration of the competing mammalian tRNA, the rate of exchange of the E. coli initiator tRNA^Met is enhanced, due to its faster rate of dissociation from the transient complex. Finally, it has been verified that in the case of the trypsin-modified methionyl-tRNA synthetase which cannot provide more than one binding site for tRNA, exchange of enzymebound bacterial tRNA by mammalian tRNA does proceed to a limiting rate independent of the mammalian tRNA concentration present in the solution.     

Selective modification of cytidine, uridine, guanosine and pseudouridine residues in Escherichia coli leucine transfer ribonucleic acid

The specificity of methoxyamine for the cytidine residues in an Escherichia coli leuoine transfer RNA (tRNA₁^leu is described in detail. Of the six non-hydrogen-bonded cytidine residues in the clover-leaf model of this tRNA, four are very reactive (C-35, 53, 85 and 86) and two are unreactive (C-67 and 79).The specificity of l-cyclohexyl-3-[2-morpholino-(4)-ethyl]carbodiimide methotosylate for the uridine, guanosine and pseudouridine residues in the leucine tRNA was also investigated. The carbodiimide completely modified four uridine residues (U-33, 34, 50 and 51) and partially modified G-37 and Ψ-39. For technical reasons, the sites of partial modification in loop I of the tRNA were difficult to establish. There was no modification of base residues in loop IV nor of U-59 at the base of stem e of the tRNA.The modification patterns described for the leucine tRNA are compared with those observed for the E. coli initiator tRNA₁^met and su⁺_III tyrosine tRNA. Several general conclusions regarding tRNA conformation are made. In particular, the evidence supporting a diversity of anticodon loop structures amongst tRNAs is discussed.     

The archaeal transamidosome for RNA-dependent glutamine biosynthesis

Archaea make glutaminyl-tRNA (Gln-tRNA^Gln) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA^Gln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA^Gln. Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA^Asn) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228–239.]. Here, we show a similar complex for Gln-tRNA^Gln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA^Gln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K_D = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA^Gln formation, with GatDE reducing the affinity of ND-GluRS for tRNA^Glu by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA^Gln) formation, and has no major effect on the kinetics of tRNA^Gln glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.     

Temperature dependence of the aminoacylation of tRNA by Bacillus stearothermophilus aminoacyl-tRNA synthetases

Valine specific transfer RNA (tRNA^Val) was isolated from Bacillus stearothermophilus and Escherichia coli by chromatography on benzoylated DEAE–cellulose (BD–cellulose). Likewise isoleucine specific transfer RNA (tRNA^Ile) was isolated from B. stearothermophilus and from Mycoplasma sp. Kid. The thermal denaturation profiles (melting curves) of the two tRNA^Val species in the presence of Mg^{+ +} were nearly identical. However, the T_m for the Kid tRNA^Ile was about 10°C lower than that for the B. stearothermophilus tRNA^Ile. A nuclease and tRNA-free aminoacyl-tRNA synthetase (AA-tRNA synthetase) preparation from B. stearothermophilus was able to function efficiently at temperatures up to 80°C in the aminoacylation of all four tRNA species. Determination of the amino acid-acceptor activity of each tRNA species as a function of temperature of the aminoacylation reaction showed in each case a strong correlation between the loss of acceptor activity and the thermal denaturation profile of the tRNA. Evidence is presented that the loss in acceptor activity is most likely due to a change in structure of the tRNA as opposed to denaturation of the enzyme. These results further support the idea that correct secondary and/or tertiary structure must be maintained for tRNA to be active as a substrate for the AA-tRNA synthetase.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Neutron scattering studies of escherichia coli tyrosyl-trna synthetase and of its interaction with trna tyr

Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of M_r, 91 (±6) × 10³ with a radius of gyration R_G of 37.8 ± 1.1 Å.The increase in the scattering mass of the enzyme upon binding tRNA^Tyr has been followed in 20 mm-imidazole · HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol, 150 mm-KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The R_G of the complex is equal to 41 ± 1 Å. Titration experiments in 74% ²H₂O, close to the matching point of tRNA, show an R_G of 38.5 ± 1 Å for the enzyme moiety in the complex. From these values, a minimum distance of 49 Å between the centre of mass of the bound tRNA and that of the enzyme was calculated.In low ionic strength conditions (20 mm-imidazole-HCl (pH 7.6), 10 mm-MgCl₂, 0.1 mm-EDTA, 10 mm-2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNA^Tyr are characterized by the appearance of aggregates, with a maximum scattered intensity at a stoichiometry of one tRNA per two enzyme molecules. At this point, the measured M_r and R_G values are compatible with a compact 1:2, tRNA: enzyme complex. This complex forms with a remarkably high stability constant: (enzyme:tRNA:enzyme)/(enzyme:tRNA)(enzyme) of 0.1 to 0.3(× 10⁶) m^?1 (at 20 °C). Upon addition of more tRNA, the complex dissociates in favour of the 1:1, enzyme:tRNA complex, which has a higher stability constant (1 to 3 (× 10⁶) m^?1).     

The Methyl Group of the N6-Methyl-N6-Threonylcarbamoyladenosine in tRNA of Escherichia coli Modestly Improves the Efficiency of the tRNA

tRNA species that read codons starting with adenosine (A) contain N⁶-threonylcarbamoyladenosine (t⁶A) derivatives adjacent to and 3′ of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA_GGU^Thr1 and tRNA_GGU^Thr3) have the t⁶A derivative N⁶-methyl-N⁶-threonylcarbamoyladenosine (m⁶t⁶A37). We have isolated a mutant of E. coli that lacks the m⁶t⁶A37 in these two tRNA_GGU^Thr species. These tRNA species in the mutant are likely to have t⁶A37 instead of m⁶t⁶A37. We show that the methyl group of m⁶t⁶A37 originates from S-adenosyl-l-methionine and that the gene (tsaA) which most likely encodes tRNA(m⁶t⁶A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA_GGU^Thr to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m⁶t⁶A37-containing tRNA_GGU^Thr species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m⁶t⁶A37 in tRNA_GGU^Thr slightly reduces the efficiency of this tRNA to read cognate codon ACC.All tRNA species from the three domains, Archaea, Bacteria, and Eucarya, contain modified nucleosides, which are derivatives of the four nucleosides, adenosine, guanosine, cytidine, and uridine. At present, more than 79 different modified nucleosides from the tRNA of various organisms have been characterized (23). Some of these are present in tRNA from only one domain, but a few are present in the same subset of and at the same position in the tRNAs from all three domains (3). One such conserved group of modified nucleosides is the threonylated adenosine (t⁶A) derivatives. These modified adenosines are present adjacent to and 3′ of the anticodon (position 37) in the subset of tRNAs that reads codons starting with A. The universal presence of t⁶A derivatives suggests that these kinds of modifications may have been present in the tRNA of the progenitor, unless a convergent evolution has occurred. This conservation also suggests that the functions of these modified nucleosides may be principally the same in all organisms.In Escherichia coli, the t⁶A37 derivative N⁶-methyl-N⁶- threonylcarbamoyladenosine (m⁶t⁶A37) is present in only two tRNA species, the tRNA_GGU^Thr species, with the same anticodon (20). Threonine is the precursor in the synthesis of t⁶A (10, 32), and in vitro threonylation requires carbonate and ATP (15, 21). Here we show that the methyl group of m⁶t⁶A37 originates from methionine. So far, no mutant deficient in any t⁶A37 derivative has been characterized. As a first step to elucidate the syntheses of these groups of modified nucleosides and their roles in vivo, we have isolated and characterized a mutant deficient in the synthesis of m⁶t⁶A37. We show that the tsaA gene most likely encodes the tRNA(m⁶t⁶A37)methyltransferase that transfers a methyl group from S-adenosylmethionine (AdoMet) to the two tRNA_GGU^Thr species containing the t⁶A moiety. The tsaA gene was localized to the 4.6 min site on the E. coli chromosome. We also show that the methyl group of m⁶t⁶A37 slightly improves the translational efficiency of the two tRNA_GGU^Thr species.     

Intrinsic Precision of Aminoacyl-tRNA Synthesis Enhanced through Parallel Systems of Ligands

MOST aminoacyl-tRNAs possessed by an organism must contain amino-acids matched to their correct anticodon so that the meaning of structural genetic information will be preserved. It is only recently, however, that we have begun to understand the underlying molecular mechanisms with regard to isoleucyl-tRNA of Escherichia coli B. Isoleucyl-tRNA synthetase, which is representative of many others in size and other properties¹, is quite selective among tRNAs, in that it binds strongly only to the cognate tRNA^Ile species². However, a weaker, but still significant affinity for non-cognate tRNAs from E. coli can be detected³. In addition, non-cognates are isoleucylated (ref. 3 and unpublished work), albeit at a maximum rate considerably slower than tRNA^Ile. Two of these reactions, the binding and isoleucylation of tRNA^phe (E. coli)³ and of tRNA_f^Met (E. coli), have been studied in detail. The generality of this phenomenon could prove important. First, the tRNA concentrations in E. coli are high (they can be no less than about 0.5 × 10^?5 M^4,5 for individual species) compared with those usual in vitro and thus even weak binding could be significant. Second, many incorrect interactions are possible. Assuming that there are about sixty molecular species of tRNA and twenty species of aminoacyl-tRNA synthetase, there are 1,200 possible interacting pairs and only about sixty of these (or 5%) are cognates. Since it is presumably desirable that misacylated tRNAs be held to a very small fraction of the total, misacylation could be significant, even if it is always a slow reaction. I have, as of writing, examined five species of purified tRNA, of which the two already mentioned give misacylations which are sufficiently facile to be easily studied under usual in vitro conditions. The other three are much less easily isoleucylated, but also give indications of reaction (my unpublished data). Thus, this limited survey emphasizes that these reactions may be common and that rejection of non-cognate tRNAs by the aminoacyl-tRNA synthetase may not be the only mechanism by which the correctness of the aminoacyl-tRNAs is assured. In fact, I have already reported^3,6 that Ile-tRNA^phe, synthesized by isoleucyl-tRNA synthetase, is rapidly destroyed by phenylal-anyl-tRNA synthetase and have suggested⁶ that the aminoacyl-tRNA synthetases may have a function in addition to synthesis of aminoacyl-tRNAs; that of destruction of misacylated cognate tRNAs.     

Kinetic Origin of Substrate Specificity in Post-Transfer Editing by Leucyl-tRNA Synthetase

The intrinsic editing capacities of aminoacyl-tRNA synthetases ensure a high-fidelity translation of the amino acids that possess effective non-cognate aminoacylation surrogates. The dominant error-correction pathway comprises deacylation of misaminoacylated tRNA within the aminoacyl-tRNA synthetase editing site. To assess the origin of specificity of Escherichia coli leucyl-tRNA synthetase (LeuRS) against the cognate aminoacylation product in editing, we followed binding and catalysis independently using cognate leucyl- and non-cognate norvalyl-tRNA^Leu and their non-hydrolyzable analogues. We found that the amino acid part (leucine versus norvaline) of (mis)aminoacyl-tRNAs can contribute approximately 10-fold to ground-state discrimination at the editing site. In sharp contrast, the rate of deacylation of leucyl- and norvalyl-tRNA^Leu differed by about 10⁴-fold. We further established the critical role for the A76 3′-OH group of the tRNA^Leu in post-transfer editing, which supports the substrate-assisted deacylation mechanism. Interestingly, the abrogation of the LeuRS specificity determinant threonine 252 did not improve the affinity of the editing site for the cognate leucine as expected, but instead substantially enhanced the rate of leucyl-tRNA^Leu hydrolysis. In line with that, molecular dynamics simulations revealed that the wild-type enzyme, but not the T252A mutant, enforced leucine to adopt the side-chain conformation that promotes the steric exclusion of a putative catalytic water. Our data demonstrated that the LeuRS editing site exhibits amino acid specificity of kinetic origin, arguing against the anticipated prominent role of steric exclusion in the rejection of leucine. This feature distinguishes editing from the synthetic site, which relies on ground-state discrimination in amino acid selection.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Crystal Structures of the Human and Fungal Cytosolic Leucyl-tRNA Synthetase Editing Domains: A Structural Basis for the Rational Design of Antifungal Benzoxaboroles

Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3′ end of tRNA^leu isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNA^leu. We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3′ adenosine of tRNA^leu at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 Å resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3′ adenosine of tRNA^leu. The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity.     

Modified nucleosides and the chromatographic and aminoacylation behavior of tRNAIle from Escherichia coli C6

Transfer RNA from Escherichia coli C6, a Met⁻, Cys⁻, relA⁻ mutant, was previously shown to contain an altered tRNA^Ile which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676–7683). We now report the purification of this altered tRNA^Ile and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA^Ile purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA^Ile (from cysteine-starved cells) was found to have a 5-fold increased V_max in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl ∼ AMP · Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA^Ile bound more efficiently to its synthetase compared to normal tRNA^Ile. Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA^Ile contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA^Ile contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA^Ile without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA^Ile has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA^Ile from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA^Ile for aminoacylation is discussed.     

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.     

Comparison of free and ribosome-bound phenylalanyl-tRNA synthetase from rabbit reticulocytes.

Phenylalanyl-tRNA synthetase (l-phenylalanine:tRNA ligase [AMP], EC 6.1.1.b) from the ribosomal and the postribosomal cell supernatant fractions of rabbit reticulocytes were purified separately and characterized. Phenylalanyl-tRNA synthetase from the ribosomal fraction was purified 114-fold to a final specific activity of 1603 units/mg and is approximately 90% pure. Phenylalanyl-tRNA synthetase from the postribosomal supernatant fraction was purified 4186-fold to a final specific activity of 247 units/mg. The enzymes from the two fractions appear to be identical based on their elution from various chromatographic media, sedimentation coefficient, pH, Mg²⁺, and K⁺ optima, and heat stability. Phenylalanyl-tRNA synthetase from rabbit reticulocytes has a molecular weight of approximately 245,000 with an α₂β₂ subunit structure. The molecular weights of the subunits are 57,000 and 67,200.     

Threonine tRNAs and their genes in Escherichia coli

Summary The subject of this study was the threonine isoacceptor family of tRNAs in Escherichia coli and the genes coding for them. The goal was to identify and map all the genes and to determine the relative contribution of each gene to the tRNA pool. The mapping experiments exploited gene-dosage effects in partially diploid strains; if a strain harboring a particular F episome overproduced a particular tRNA species, it could be concluded that the gene for that tRNA was located on the chromosomal segment carried by the F. Isoacceptor tRNAs were distinguished by column fractionation. It was found that there are three major threonine tRNA species that occur in roughly equal amounts. These are tRNA ₁ ^Thr , which is encoded by a gene in the distal region of the rrnD ribosomal RNA operon, and tRNA ₃ ^Thr and tRNA ₄ ^Thr , which come from genes in the cluster thrU tyrU glyT thrT at 89 min on the map. The relative abundances of the tRNA species roughly match the reported frequencies of the codons that they recognize in mRNA. Although the tRNA ₄ ^Thr has a mismatched base pair that raised questions about its biological activity, it was found to be functional at least with respect to recognition by the threonyl-tRNA synthetase. An apparent fourth gene affecting threonine tRNA has been identified and mapped at 6–8 min; it is here designated thrW. It may be a structural gene for a minor tRNA species, present in one-third the amount of each of the major species, and chromatographically indistinguishable from tRNA ₄ ^Thr .A preliminary report of most of this work has appeared previously (M.M. Comer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, p. 109)     

Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA^His have distinctive identity elements, we constructed E. coli tRNA^His _CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA^His _CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA^His _CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA^His _CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.     

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.