Yeast aminoacyl-tRNA Synthetases Research Articles

Small-angle X-ray Solution Scattering Study of the Multi-aminoacyl-tRNA Synthetase Complex Reveals an Elongated and Multi-armed particle

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Structural basis of yeast aminoacyl-tRNA synthetase complex formation revealed by crystal structures of two binary sub-complexes

The yeast aminoacyl-tRNA synthetase (aaRS) complex is formed by the methionyl- and glutamyl-tRNA synthetases (MetRS and GluRS, respectively) and the tRNA aminoacylation cofactor Arc1p. It is considered an evolutionary intermediate between prokaryotic aaRS and the multi- aaRS complex found in higher eukaryotes. While a wealth of structural information is available on the enzymatic domains of single aaRS, insight into complex formation between eukaryotic aaRS and associated protein cofactors is missing. Here we report crystal structures of the binary complexes between the interacting domains of Arc1p and MetRS as well as those of Arc1p and GluRS at resolutions of 2.2 and 2.05 Å, respectively. The data provide a complete structural model for ternary complex formation between the interacting domains of MetRS, GluRS and Arc1p. The structures reveal that all three domains adopt a glutathione S-transferase (GST)-like fold and that simultaneous interaction of Arc1p with GluRS and MetRS is mediated by the use of a novel interface in addition to a classical GST dimerization interaction. The results demonstrate a novel role for this fold as a heteromerization domain specific to eukaryotic aaRS, associated proteins and protein translation elongation factors.

Structural basis of yeast aminoacyl-tRNA synthetase complex formation

Bcl-2 family proteins play a central role in regulating the intrinsic apoptotic pathway. Pro-survival family members such as Bcl-2, Bcl-xL, Bcl-w,Mcl-1 andA1 protect cells from apoptosis. Pro-apoptotic BH3-only proteins such as Bim, Puma, Noxa, Bad, Bmf, and Bid promote apoptosis by interacting with and inactivating pro-survival proteins. It was originally thought that each pro-apoptotic BH3-only protein could interact with any pro-survival protein. However, it has recently become clear that there is selectivity within the pathway suggesting functional groupings [1]. Bim and Puma behave as originally predicted and can interact with all pro-survival proteins and are potent killers. In contrast, Noxa and Bad interact with distinct subsets of pro-survival proteins. Noxa only interacts with Mcl-1 and A1, while Bad interacts with Bcl-2, Bcl-xL and Bcl-w. As a result, either Noxa or Bad acting alone is a weak killer, but together they are potent. Other BH3-only proteins are not as promiscuous as Bim and Puma, but not as selective as Noxa or Bad. This work aims to structurally characterise interactions between A1 and various BH3-domain peptides, enabling the determinants of selective binding to be identified. Crystals have been obtained of A1 in complex with several BH3-domain peptides. Progress on structural characterisation will be reported.

Arc1p Organizes the Yeast Aminoacyl-tRNA Synthetase Complex and Stabilizes Its Interaction with the Cognate tRNAs

Eukaryotic aminoacyl-tRNA synthetases, in contrast to their prokaryotic counterparts, are often part of high molecular weight complexes. In yeast, two enzymes, the methionyl- and glutamyl-tRNA synthetases associate in vivo with the tRNA-binding protein Arc1p. To study the assembly and function of this complex, we have reconstituted it in vitro from individually purified recombinant proteins. Our results show that Arc1p can readily bind to either or both of the two enzymes, mediating the formation of the respective binary or ternary complexes. Under competition conditions, Arc1p alone exhibits broad specificity and interacts with a defined set of tRNA species. Nevertheless, the in vitro reconstituted Arc1p-containing enzyme complexes can bind only to their cognate tRNAs and tighter than the corresponding monomeric enzymes. These results demonstrate that the organization of aminoacyl-tRNA synthetases with general tRNA-binding proteins into multimeric complexes can stimulate their catalytic efficiency and, therefore, offer a significant advantage to the eukaryotic cell.

Accuracy of Protein Biosynthesis: Quasi-species Nature of Proteins and Possibility of Error Catastrophes

Yeast aminoacyl-tRNA synthetases act in a multi-step process when recognizing their cognate amino acids; this identification event includes “physical” binding and “chemical” proof-reading steps. However, the various enzymes use these single steps at different degrees, and their specificities with regard to the 20 naturally occurring amino acids deviate considerably from each other. The characteristic discrimination factors D were determined for seven synthasesin vitro: the highest specificity withDvalues between 28000 and >500000 were observed with tyrosyl-tRNA synthetase, the lowest values between 130 and 1700 for lysyl-tRNA synthetase. The tested class I enzymes are more specific than the investigated class II enzymes, and it may be put into discussion whether this observation can be generalized.Error rates in amino acid recognition differ not only between the individual aminoacyl-tRNA synthetases but also considerably for different amino acids sorted by the same enzyme. Strikingly, all investigated enzymes exhibit a poor specificity in discrimination of cysteine and tryptophan from their cognate substrates, and these cases may be regarded as “specificity holes”.In view of the observed specificities a protein consisting of 700 amino acids would contain maximally up to five “incorrect” residues, if thein vitroerror rates are also valid underin vivoconditions. Therefore the terminus “quasi-species”, an expression which was originally created for nucleic acids, is justified. The “quasi-species” nature of proteins may become important when genes are translated in different organisms with different accuracies of the translation apparatus. In such cases different “quasi-species” will be obtained.Using our data in mathematical models which predict the stability of protein synthesizing systems, we find that they are consistent with a stable yeast organism which is not prone to die by an [puod]error catastrophe”. However, this appears only if average values from our experiments are used for calculations. If a single compound, e.g. the arginine analog canavanine, is discriminated very poorly from the cognate substrate, or if the “specificity holes” get larger, an “error catastrophe” must be envisaged.

Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm?

The aminoacyl-tRNA synthetases from a crude extract of yeast were shown to bind to heparin-Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not. The behaviour of purified lysyl-tRNA synthetases from yeast and E. coli was examined in detail. The native dimeric enzyme from yeast (Mr 2 X 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E. coli (Mr 2 X 65000) displayed no affinity for these supports. Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 X 65500. A structural model is proposed, according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a 'binding' domain responsible for association to negatively charged carriers. The evolutionary acquisition of this property by lower eukaryotic aminoacyl-tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis. We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.

Comparative study of the effect of ochratoxin a analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5 ' chlorinated dihydromethyl isocoumarin linked to l,ß-phenylalanine by an α-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin α (OTα) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OTα were ineffective.

Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver

The subcellular distribution of five aminoacyl-tRNA synthetase from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.

Protein fingerprinting by SDS-gel electrophoresis after partial fragmentation with CNBr

The CNBr fragmentation reaction was adapted for the degradation of microgram quantities of protein in sodium dodecyl sulfate gel bands. These bands are then electrophoresed so as to generate easily distinguishable protein fingerprints. The method was tested on three proteins of known sequence and shown to generate nearly the full range of possible peptide fragments. It was also employed to compare the structures of several yeast aminoacyl tRNA synthetases and a contaminating crystallizable protein.

The yeast aminoacyl-tRNA synthetases: Methodology for their complete or partial purification and comparison of their relative activities under various extraction conditions

Several fractionation steps are described which can be applied to the partial purification of the 20 aminoacyl-tRNA synthetases from commercial baker's yeast. Comparative experiments performed in the presence or absence of protease inhibitors revealed that some enzymes prepared in the presence of the inhibitor exhibit much higher specific activities than the proteins extracted in the absence of the inhibitor. The methodology reported can be used for the simultaneous preparation of several pure aminoacyl-tRNA synthetases. As examples, the large scale purification of phenylalanyl-and valyl-tRNA synthetases are described.

Site of aminoacylation of tRNAs from Escherichia coli with respect to the 2'- or 3'-hydroxyl group of the terminal adenosine.

A method is presented by which the site of primary attachment of the amino acids with respect to the 2'- or 3'-hydroxyl group of the terminal adenosine of E. coli tRNAs can be determined. It is found that the aminoacyl-tRNA synthetases (EC 6.1.1.-) with specificity for Arg, Asn, Ile, Leu, Met, Phe, Thr, Trp, and Val attach the amino acid to the 2'-position; those with specificity for Gly, His, Lys, and Ser attach the amino acid to the 3'-position; and that Tyr and Cys can be enzymatically attached to both the 2'- and 3'-positions. Together with previous experiments on yeast aminoacyl-tRNA synthetases, it is now shown that the specificity for one particular hydroxyl group is preserved during the evolution from prokaryotic to eukaryotic systems.

Differences in the Thermal Inactivation Properties of Lysyl and Arginyl Transfer Ribonucleic Acid Synthetases of Bakers' Yeast

Abstract An investigation was made of the thermal inactivation of lysyl-tRNA synthetase (l-lysine:tRNA ligase (AMP), EC 6.1.1.6) 400-fold purified from bakers' yeast. During heat inactivation at pH 7.2, 45°, the enzyme was stabilized by the presence of l-lysine or by ATP and MgCl2. The concentration of l-lysine giving half-maximal protection of the enzyme was decreased by the presence of ATP and MgCl2, or tRNA and MgCl2. Similarly, polyuridylate and polycytidylate enhanded the protection of the synthetase by l-lysine. No stabilization was given by any polynucleotide in the absence of l-lysine. Adenosine 5'-phosphate, adenosine 2'(3')-monophosphate, adenosine, 5-hydroxy-dl-lysine, and ethylene glycol each gave some stabilization of the enzyme in certain conditions. In a comparative study it was found that partially purified preparations of arginyl-tRNA synthetase (l-arginine:tRNA ligase (AMP), EC 6.1.1) from bakers' yeast showed a different pattern of susceptibility to heat from that shown by the lysine enzyme. Low concentrations of l-arginine were less effective than tRNA and MgCl2 in stabilization of arginyl-tRNA synthetase from thermal inactivation at 50°. The greatest protection was observed in the presence of l-arginine, tRNA, and MgCl2, whereas l-arginine, ATP, and MgCl2 gave lower protection. The characteristics of heat inactivation of the yeast aminoacyl-tRNA synthetases specific for lysine and arginine could be interpreted in terms of substrate-induced conformational changes in the enzyme proteins.

Studies on Polynucleotides: XCI. Yeast Methionine Transfer Ribonucleic Acid: Purification, Properties, and Terminal Nucleotide Sequences

Abstract Countercurrent distribution of baker's yeast transfer RNA (tRNA) gives rise to two peaks (I and II) of methionine acceptor activity. Methionine tRNA (tRNAfmet) (Peak I) has been purified to homogeneity by ion exchange chromatography on DEAE-Sephadex and by reverse phase chromatography. It has the following properties. (a) It can be aminoacylated with methionine by either crude yeast or Escherichia coli aminoacyl tRNA synthetases. (b) The methionyl-tRNA thus formed can be further converted to N-formyl-methionyl-tRNA (fMet-tRNAfmet) in the presence of formyl tetrahydrofolic acid and E. coli methionyl-tRNA transformylase. (c) Amino acid incorporation studies in vitro with poly r(U-G) and poly r(A-U-G) as messengers show that, similar to E. coli fMet-tRNAfmet, the yeast fMet-tRNAfmet can bring about the initiation of protein synthesis under the direction of either of the initiator codons G-U-G or A-U-G. Analysis of fragments produced by the action of takadiastase Ribonuclease T1 show that the 5'- and 3'-terminal nucleotide sequences of yeast tRNAfmet are pA-G-, and C-U-A-C-C-A, respectively; the terminal sequences thus derived are not identical with those present in E. coli tRNAfmet. Under conditions of magnesium ion concentration (5 mm) used for the aminoacylation of tRNAfmet (Peak I), tRNAmet (Peak II) is aminoacylated by crude E. coli aminoacyl tRNA synthetases at a slow rate. At 15 mm Mg++, however, tRNAmet (Peak II) is aminoacylated with methionine to the same extent by crude E. coli aminoacyl tRNA synthetases as by yeast aminoacyl tRNA synthetases. Met-tRNA from Peak II cannot be converted to fMet-tRNA by E. coli extracts.

Characterization of Two Species of Methionine Transfer Ribonucleic Acid from Bakers' Yeast

Abstract The biochemical properties of two species of methionine transfer ribonucleic acid from bakers' yeast, separated by diethylaminoethyl Sephadex column chromatography, have been studied. Each species of the two methionine tRNAs, designated methionine tRNA I and methionine tRNA II, respectively, has been purified by benzoylated DEAE-cellulose column chromatography. Methionine tRNA I can be esterified with methionine by Escherichia coli aminoacyl-tRNA synthetase as efficiently as by yeast aminoacyl-tRNA synthetase, whereas methionine tRNA II can be charged only partially by the E. coli enzyme under the same conditions used in the charging of methionine tRNA I. Methionine tRNA I has been shown to be converted to N-formylmethionyl-tRNA I by E. coli methionyl-tRNA transformylase in the presence of N10-formyltetrahydrofolate, while methionine tRNA II is not. In ribosomal binding studies, 14C-methionyl-tRNA I is recognized by the condons AUG, GUG, and UUG, while 14C-methionyl-tRNA II responds principally to AUG. In addition, methionine has been found to be incorporated from both methionyl-tRNAs I and II into polypeptide as directed by messenger RNA obtained from bacteriophage f2 in a cell-free system from E. coli. Our present report also provides evidence that yeast N-formylmethionyl-tRNA I serves as an initiator of protein synthesis programmed with the natural messenger in an E. coli system in vitro.