Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of the cytoplasmic and chloroplastic enzymes of a higher plant (Phaseolus vulgaris)

The phenylalanyl-tRNA synthetases from cytoplasm and chloroplasts of bean (Phaseolus vulgaris) leaves employ different strategies with respect to accuracy. The chloroplastic enzyme that is coded for by the nuclear genome follows the pathway of posttransfer proofreading, also characteristic for enzymes from eubacteria and cytoplasm and mitochondria of lower eukaryotic organisms. In contrast, the cytoplasmic enzyme uses pretransfer proofreading in the case of noncognate natural amino acids, characteristic for higher eukaryotic organisms and archaebacteria. Dependent on the nature of the noncognate amino acid, pretransfer proofreading in this case occurs without tRNA stimulation or with tRNA stimulated with no or little effect of the nonaccepting 3'-OH group of the terminal adenosine. The fundamental mechanistic difference in proofreading between the heterotopic intracellular isoenzymes of the plant cell supports the idea of the origin of the chloroplastic gene by gene transfer from a eubacterial endosymbiont to the nucleus. Origin by duplication of the nuclear gene, as indicated for mitochondrial phenylalanyl-tRNA synthetases [Gabius, H.-J., Engelhardt, R., Schroeder, F.R., & Cramer, F. (1983) Biochemistry 22, 5306-5315], appears unlikely. Further analyses of the ATP/PPi pyrophosphate exchange and aminoacylation of tRNAPhe-C-C-A(3'NH2), using 11 phenylalanine analogues, reveal intraspecies and interspecies variability of the architecture of the amino acid binding part within the active site.     

Diadenosine oligophosphates: peculiarities of synthesis by phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8.

Temperature and other factors affecting synthesis of bis(5'-adenosyl) tetraphosphate (Ap4A) and bis(5'-adenosyl)triphosphate (Ap3A) catalyzed by phenylalanyl-tRNA synthetases (PheRSs) from Escherichia coli MRE-600 and Thermus thermophilus HB8 have been investigated. Those two synthetases exhibited different temperature-dependent rates of the Ap4A and Ap3A synthesis. However, with respect to the effects of such effectors of the Ap4A synthesis as Zn2+, Mg2+, tRNA and Ap4A phosphonate analogues, as well as some inhibitors of aminoacyl-tRNA synthetase, those two enzymes were apparently undistinguishable.     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.     

Effect of diadenosine oligophosphates (Ap4A and Ap3A) and their phosphonate analogs on catalytic properties of phenylalanyl-tRNA synthetase from E. coli

The influence of P1,P3-bis(5'-adenosyl)triphosphate (Ap3A), P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and its analogues, containing a residue of methylenediphosphonic acid in various positions of the oligophosphate chain, on the reactions catalysed by phenylalanyl-tRNA synthetase from E. coli MRE-600 has been studied. The compounds do not affect significantly the rate of ATP-[32P]PPi-exchange nor maintain this reaction in the absence of ATP. The diadenosineoligophosphates are shown to be noncompetitive inhibitors of ATP in the tRNA aminoacylation by phenylalanine (for Ap4A Ki = 1,45.10(-3) M). The phosphonate analogues of Ap4A inhibit the synthesis of Ap3A depending on their structure. The conclusion is thus drawn that the E. coli MRE-600 phenylalanyl-tRNA synthetase does not interact property with Ap4A and its phosphonate analogues.     

Structural basis for discrimination of L-phenylalanine from L-tyrosine by phenylalanyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) exert control over the faithful transfer of amino acids onto cognate tRNAs. Since chemical structures of various amino acids closely resemble each other, it is difficult to discriminate between them. Editing activity has been evolved by certain aaRSs to resolve the problem. In this study, we determined the crystal structures of complexes of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with L-tyrosine, p-chloro-phenylalanine, and a nonhydrolyzable tyrosyl-adenylate analog. The structures demonstrate plasticity of the synthetic site capable of binding substrates larger than phenylalanine and provide a structural basis for the proofreading mechanism. The editing site is localized at the B3/B4 interface, 35 A from the synthetic site. Glubeta334 plays a crucial role in the specific recognition of the Tyr moiety in the editing site. The tyrosyl-adenylate analog binds exclusively in the synthetic site. Both structural data and tyrosine-dependent ATP hydrolysis enhanced by tRNA(Phe) provide evidence for a preferential posttransfer editing pathway in the phenylalanine-specific system.     

On the stereochemistry of activation of phenylalanine by phenylalanyl-tRNA synthetase from baker's yeast.

Because of its chiralic alpha-phosphorus atom adenosine 5'-O-(1-thiotriphosphate) (ATPalphaS) exists in two diastereomeric forms, arbitrarily named (A) and (B). For phenylalanyl-tRNA synthetase ATPalphaS (A) is a substrate whereas ATPalphaS (B) is neither a substrate nor an inhibitor. During the ATPalphaS (A)/PPi exchange reaction with phenylalanyl-tRNA synthetase the configuration at the alpha-phosphorus is retained. The mechanistic implications of these findings are discussed. Preliminary investigations with several other aminoacyl-tRNA synthetases show that the stereochemical requirement with respect to the alpha-phosphorus of ATP is not identical for all aminoacyl-tRNA synthetases.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate by organellar and cytoplasmic phenylalanyl-tRNA synthetases of Euglena gracilis

Purified phenylalanyl-tRNA synthetases present in chloroplasts, mitochondria and cytoplasm of green and bleached Euglena gracilis strains, respectively, are able to synthesize diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A). Ap4A synthesis is strictly dependent on zinc ions. This is the first evidence that chloroplasts should be able to synthesize Ap4A. Synthesis of Ap4A by phenylalanyl-tRNA synthetases of the three compartments of a plant cell or by other enzymes such as Ap4A phosphorylase is discussed.     

Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.     

Yeast phenylalanyl-tRNA synthetase. Properties of the histidyl residues.

Reactivity of the histidyl groups of yeast phenylalanyl-tRNA synthetase was studied in the absence or presence of substrates. In the absence of substrates about 10 histidine residues were found to react with similar kinetic constants. Phenylalanine at 10(-3) M was found to protect two histidyl residues; increasing the amino acid concentration to 5 . 10(-3) M resulted in the protection of two more histidyl groups. tRNAPhe did not afford any protection to histidine residues, but acylated phenylalanyl-tRNA (Phe-tRNAPhe) protected two of the four histidyl groups already protected by phenylalanine. These results suggest the existence of two different sets of accepting sites for phenylalanine: one specific for the free amino acid, the other one specific for the amino acid linked to the tRNA, but being accessible to free phenylalanine, with a somewhat lower binding constant, ATP was found to mask around four histidyl residues against diethylpyrocarbonate modification. By photoirradiation of enzyme-phenylalanine complex in the presence of rose bengale, a significant amount of amino acid was bound to the alpha subunit (Mr = 73 000) of phenylalanyl-tRNA synthetase, confirming that the amino acid binding site is located on this subunit, as previously suggested by modification of thiol groups. Upon irradiation of an enzyme-tRNA complex, almost no covalent binding of tRNA occurred during enzyme inactivation, suggesting that the histidyl residues involved in the enzymic activity are not required for tRNA binding.     

Prokaryotic and eukaryotic tetrameric phenylalanyl-tRNA synthetases display conservation of the binding mode of the tRNA(Phe) CCA end

The interaction of human phenylalanyl-tRNA synthetase, a eukaryotic prototype with an unknown three-dimensional structure, with the tRNA(Phe) acceptor end was studied by s(4)U-induced affinity cross-linking with human tRNA(Phe) derivatives site-specifically substituted at the single-stranded 3' end. Two different subunits of the enzyme bind two adjacent nucleotides of the tRNA(Phe) 3' end: nucleotide 76 is associated with the catalytic alpha subunit, while nucleotide 75 is in contact with the beta subunit. The binding mode is similar to that revealed previously in structural and affinity cross-linking studies of the prokaryotic Thermus thermophilus phenylalanyl-tRNA synthetase. Our results suggest that the distinctive features of tRNA(Phe) acceptor end binding are conserved for the eukaryotic and prokaryotic tetrameric phenylalanyl-tRNA synthetases despite their significant differences in the domain composition of the beta subunits. The data from affinity cross-linking experiments with human phenylalanyl-tRNA synthetase complexed with small ligands (ATP and/or phenylalanine or a stable synthetic analogue of phenylalanyl adenylate) reveal that the location of the tRNA(Phe) acceptor end varies with the presence and nature of other substrates. The lack of substrate activity of human tRNA(Phe) substituted with s(4)U at the 3'-terminal position suggests that base-specific interactions of the terminal adenosine are critically important for a productive interaction. The conformational rearrangement of the tRNA 3' end induced by the other substrates and dictated by base-specific contacts of the terminal nucleotide is an additional means of ensuring the phenylalanylation specificity in both prokaryotic and eukaryotic systems.     

Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.     

The tRNA-induced conformational activation of human mitochondrial phenylalanyl-tRNA synthetase

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.     

Molecular and functional properties of an archaeal phenylalanyl-tRNA synthetase from the hyperthermophile Sulfolobus solfataricus

An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile Sulfolobus solfataricus (Ss). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, SsFRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. SsFRS interacts with the elongation factor 1alpha isolated from the same source; this caused a significant enhancement of the SstRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal SsFRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.     

Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA.

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.     

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus

FPLC separation of - and β-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native -subunits of both enzymes were primarily ₂-dimers and tended to aggregate. Most E. coli enzyme β-subunits were monomeric and only a small fraction was represented by β₂-dimers. All thermophilic β-subunits were β-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA^Phe aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding β-subunits. -Subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.     

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].