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PubMed1995

Threonyl-tRNA synthetase.

Threonine contributes to the solubility and reactivity of proteins by its hydroxy group as well as to the formation and stability of the hydrophobic core of proteins by its methyl group. One may assume that the use of this bifunctional and simply structured amino acid was established early in evolution. Whereas the catalytic pathway of threonine activation and transfer into protein does not deviate essentially from those catalyzed by other aminoacyl-tRNA synthetases, the enzyme specific for threonine exhibits several interesting individual properties: its biosynthesis is regulated by feedback mechanisms, it can be selectively inhibited (out of twenty aminoacyl-tRNA synthetases) by the antibiotic borrelidin, and it can be a target for autoantibodies, thus being involved in the course of autoimmune diseases. The enzyme has been isolated from more than ten organisms showing a dimeric nature and molecular masses between 110 and 220 kDa. Additionally, in several of these cases, the gene of threonyl-tRNA synthetase has been localized, cloned and sequenced, exhibiting proteins of 400 to 800 amino acids chain length. More interesting facts can be expected from future research ranging from chemistry and molecular biology to medicine, e.g. by elucidation of the three dimensional structures of threonyl-tRNA synthetases and of their antigenic epitopes, possibly followed by therapeutic use of less antigenic mutant proteins.

Animals↗
PubMed1993

Direct evidence for selective modulation of psbA, rpoA, rbcL and 16S RNA stability during barley chloroplast development.

The turnover of RNAs encoded by seven different barley chloroplast genes was analyzed after treatment of barley shoots with tagetitoxin, a selective inhibitor of chloroplast transcription. Changes in RNA stability were examined during chloroplast development using basal and apical leaf sections of 4.5-day-old dark-grown seedlings and apical leaf sections of 4.0-day-old dark-grown seedlings which had been illuminated for 12 h. Of the RNAs examined, a 2.6 kb unspliced precursor of tRNA(lys) exhibited the shortest half-life, which was estimated to be 3 h. The 16S rRNA and psbA mRNA had the longest estimated half-lives, which were greater than 40 h. Among mRNAs, half-lives were estimated to range from 6 h for psaA mRNA, to over 40 h for psbA mRNA. Therefore, barley chloroplast mRNAs have long half-lives relative to bacterial mRNAs. The stability of atpB mRNA and the unspliced precursor of tRNA-lys was not altered during chloroplast development, while the stability of psaA mRNA decreased 2-fold. In contrast, the stability of the 16S rRNA and mRNAs for rpoA, psbA and rbcL increased during chloroplast development. The stability of 16S rRNA increased markedly during chloroplast development in the dark and this increase was maintained in illuminated seedlings. The stability of rbcL mRNA increased 2.5-fold during chloroplast development in the dark, and then decreased 2-fold in chloroplasts of light-grown plants. The initial increase in rpoA and psbA mRNA stability was also light-independent, with total increases in stability of at least 5-fold. In the case of rpoA, the stability of 2 of the 13 polycistronic rpoA transcripts that were detected in dark-grown plants was selectively increased during chloroplast development. In conclusion, the stability of some transcripts is selectively increased and further modulated during chloroplast development in barley. We propose that the selective stabilization of chloroplast mRNA, which occurred independent of light, is an indication that non-light regulated developmental signals are involved in barley chloroplast mRNA stability.

Base Sequence↗
PubMed1985

Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet.

It was previously shown that when the tryptic fragment of methionyl-tRNA synthetase from Escherichia coli is incubated with periodate-treated initiator tRNA, it is inactivated due to the formation of a covalent 1:1 complex that could be stabilized by reduction with cyanoborohydride [Hountondji, C., Fayat, G., & Blanquet, S. (1979) Eur. J. Biochem. 102, 247-250]. In this work, the residues labeled in the trypsin-modified enzyme have been identified. After chymotryptic digestion of the protein-tRNA complex, two major labeled peptides (A and B) and a minor one (C) were isolated and identified by sequencing. The radioactivity associated with peptides A-C represented 65-75, 20-25, and 2-4%, respectively, of the radioactivity eluted from the peptide maps. Peptides A and B encompassed lysines-335 and -61, respectively. Both these lysines were fully labeled. Peptide C encompassed lysines-142, -147, and -149, each of which was incompletely labeled. The significance of these results is discussed in light of the known crystallographic structure of the enzyme.

Amino Acid Sequence↗
PubMed1986

Internal thermodynamics of position 51 mutants and natural variants of tyrosyl-tRNA synthetase.

Natural variation and evolution impose structural changes on an enzyme that can affect the energetics of catalysis. The energy profile of reaction could, in theory, be altered in three distinct ways: uniform binding changes, differential binding changes, and catalysis of elementary steps. Residue threonine-51 of tyrosyl-tRNA synthetase from Bacillus stearothermophilus is subject to natural variation, being replaced by alanine and proline in the enzymes from Bacillus caldotenax and Escherichia coli, respectively. The consequences of this variation on the energetics of formation of tyrosyl adenylate have been investigated by constructing free energy profiles for wild-type and mutant enzymes constructed by introducing these amino acids into the B. stearothermophilus enzyme. Mutation of Thr-51 to alanine, proline, and cysteine by site-directed mutagenesis improves the stabilization of the transition state in the formation of tyrosyl adenylate. Most marked is the mutation Thr-51----Pro-51 which stabilizes the transition state by 2.2 kcal/mol and accelerates the forward rate 20-fold to a level near that of the enzyme from E. coli. However, the improved transition-state binding is accompanied by an even greater stabilization of tyrosyl adenylate. This reduces the rate of pyrophosphorolysis of tyrosyl adenylate and/or weakens the binding of pyrophosphate in the reverse reaction, shifting the equilibrium between enzyme-bound reactants greatly in favor of the enzyme-intermediate complex. The more stable mutant enzyme-tyrosyl adenylate complexes have lower rates of aminoacylation, suggesting that mutations which stabilize the intermediate slow down the subsequent transfer of tyrosine from tyrosyl adenylate to tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Consecutive low-usage leucine codons block translation only when near the 5' end of a message in Escherichia coli.

Insertion of nine consecutive low-usage CUA leucine codons after codon 13 of a 313-codon test mRNA strongly inhibited its translation without apparent effect on translation of other mRNAs containing CUA codons. In contrast, nine consecutive high-usage CUG leucine codons at the same position had no apparent effect, and neither low- nor high-usage codons affected translation when inserted after codon 223 or 307. Additional experiments indicated that the strong positional effect of the low-usage codons could not be accounted for by differences in stability of the mRNAs or in stringency of selection of the correct tRNA. The positional effect could be explained if translation complexes are less stable near the beginning of a message: slow translation through low-usage codons early in the message may allow most translation complexes to dissociate before they read through.

Blotting, Northern↗
PubMed1995

Informational suppression to investigate structural functional and evolutionary aspects of the Erwinia chrysanthemi cellulase EGZ.

The cellulase EGZ produced by the plant pathogen Erwinia chrysanthemi belongs to family 5 of the beta-glycohydrolases (also referred to as cellulase family A), which contains over 40 members from Gram-negative and Gram-positive bacteria and fungi. Amber mutations were introduced into 16 codons of the celZ gene encoding EGZ. Targeted residues included: (1) two Glu, two His and one Arg residue, strictly conserved throughout family 5; (2) one Arg and one His residue conserved in sub-family 5-2; and (3) one His and six Arg residues not conserved at all. Each amber allele was introduced into 13 Escherichia coli strains each carrying a different suppressor tRNA that inserts an amino acid at the mutated position. In vivo stability of the mutated forms of EGZ and their cellulase activity were analysed as well as suppression efficiency. For some positions of particular interest, missense mutations were introduced into the celZ gene either to confirm the effect of the suppressor-mediated amino acid substitution or to broaden the spectrum of mutations available. The substitution patterns of the two Glu positions were interpretable in the light of the stereospecificity of the reaction catalysed by EGZ: Glu133 and Glu220 are proposed to act as a proton donor and as a nucleophile, respectively, forming the glycosyl-enzyme intermediate. Substitution at His-occupied positions, including two non-conserved positions, yielded proteins affected in their catalytic activity but not their in vivo stability. In particular, evidence was obtained for His at position 98 to be involved in interactions with the substrate. The view that Arg residues are important in stabilizing proteins was supported by the identification of three Arg residues, whose substitution yielded thermosensitive forms of EGZ. In addition, Pro substitutions of any of the six Arg residues altered protein stability in vivo but the substitutions scored almost neutral for activity. Five positions, predicted to be within alpha-helices, were found to be susceptible to Pro substitutions (but not to Ala) with respect to stability in vivo. Overall, the systematic alteration of all His and Arg residues coupled with the simultaneous analysis of activity and in vivo stability allowed us to demonstrate that substitution matrices vary at each position and for each biological property considered. Ideally, therefore, substitution matrices used in sequence alignment procedures should be reconsidered as position-specific and as property-specific.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Centromeric DNA of Kluyveromyces lactis.

A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.

Centromere↗
PubMed1983

Multienzyme complexes of eukaryotic aminoacyl-tRNA synthetases.

Eukaryotic aminoacyl-tRNA synthetases, unlike their prokaryotic counterparts, may occur as high-Mr multienzyme complexes. Recently, successful purification of synthetase complexes makes possible the elucidation of the structural organization of these high-Mr complexes. Although their physiological significance remains unknown, recent studies suggest some possible functional roles for these complexes.

Amino Acids↗
PubMed2004

Structural basis for the control of translation initiation during stress.

During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures. Furthermore, protein Y competes with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature.

Bacterial Proteins↗
PubMed1999

Dimer formation by tRNAs.

Available data on formation of dimers and other oligomers by "mature" tRNAs and the transcripts of their genes show that the dimers may be formed in vitro during the purification of individual tRNAs as well as on heating and subsequent cooling of tRNA preparations. The functional activity of dimers varies for different tRNAs. Structural analysis of tRNAs that are able or unable to form dimers suggests the importance of the hairpin melting temperature for dimer formation. Pathways of dimer formation and specific structural features of tRNA dimers are discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1994

Upstream interactions of functional mammalian tRNA gene transcription complexes probed using a heterologous DNA-binding protein.

An oligonucleotide containing the recognition site for the Escherichia coli lac repressor was inserted at various positions in the 5' flanking region of a human serine tRNA gene, and the consequences of binding lac repressor on in vitro transcription by RNA polymerase III were investigated. lac repressor prebound to operator sites centered at positions -9, -15, -35, and -37 upstream of the mature tRNA coding region completely inhibited transcription by interfering with the formation or stability of transcription complexes. lac repressor also inhibited transcription of tDNA derivatives containing operator sites at -9 and -15 when added following assembly of transcription complexes or during ongoing synthesis, but had no effects on the other tDNA derivatives if added subsequent to complex assembly. lac repressor prebound at position -43 and -46 partially inhibited transcription and redirected initiation to sites farther downstream. These effects required the continued presence of bound repressor protein. Our findings demonstrate that the human RNA polymerase III transcription complex extends at least 35 nucleotides upstream of the coding region and suggest that the spatial constraints imposed by a protein bound this far upstream can alter start site selection. Moreover, the flanking region encompassing the transcription start site remains accessible to DNA-binding proteins following assembly of the initiation complex and throughout multiple rounds of transcription.

Base Sequence↗
PubMedDate not supplied

[Interaction of deacylated phenylalanyl tRNA from yeasts with Escherichia coli ribosomes. The role of the modified nucleotide in codon-anticodon interaction].

The method of anticodon loop replacement has been used to make derivatives of yeast tRNA(Phe)GmAAY with the substitution at the 37 position (tRNA(Phe)GAAA), and at both the anticodon (tRNA(Phe)GCAG) and the 37 position. A quantitative study of the interaction of various types of yeast deacylated tRNA: tRNA(Phe)GmAAY, tRNA(Phe)GAAA, tRNA(Phe)GCAG, and tRNA(Phe)-Y with the P site of the 70S ribosome.poly(U) complex was carried out at different Mg2+ concentrations and temperatures. The replacement of the Y base on the nonmodified adenosine decreases the interaction enthalpy from 39 to 24 kcal/mole, whereas the complete removal of the Y base reduces the interaction enthalpy to 16 kcal/mole. The replacement of the second letter of the anticodon (A) with cytosine leads to a drop in the enthalpy to 6 kcal/mole, which is typical of tRNA interaction with the P site in the absence of poly(U). In the absence of poly(U) the affinity of tRNA(Phe)-Y for the P site of the 70S ribosome is 5 times lower than the affinity of tRNA(Phe)GmAAY and tRNA(Phe)GCAG. Thus, in the ribosome the modified nucleotide not only stabilizes the codon-anticodon interaction owing to the stacking interaction with the stack of codon-anticodon bases, but also lowers the free energy of binding as a result of the interaction of the modified nucleotide itself with the hydrophobic center of the P site on the ribosome.

Acylation↗
PubMed2000

An intermediate step in the recognition of tRNA(Asp) by aspartyl-tRNA synthetase.

The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.

Anticodon↗
PubMed1984

Electron microscopy study of the aminoacyl-tRNA synthetase multienzymatic complex purified from rabbit reticulocytes.

The morphology of the high molecular weight complex of aminoacyl-tRNA synthetases purified from rabbit reticulocytes has been investigated by electron microscopy. To stabilize it against dissociation, the complex was also studied after chemical crosslinking. Freeze fracture, drying shadowing and negative staining were used. The reticulocyte complex appears as a moderately elongated object with no simple compact shape. Upon rapid drying, the native complex dissociates and shows the presence of approximately 8 globular components, the individual size of which is 80-100 A. The surface of the cross-linked complex shows several distinct globules which appear to extend out of a central core. The irregularly shaped crosslinked complex has a maximal dimension of 350 +/- 50 A. The morphology of the synthetase complex is discussed with respect to some of the properties of this type of multienzymatic system.

Amino Acyl-tRNA Synthetases↗
PubMed1989

Identification of an amino acid region supporting specific methionyl-tRNA synthetase: tRNA recognition.

Site-directed nuclease digestion and nonsense mutations of the Escherichia coli metG gene were used to produce a series of C-terminal truncated methionyl-tRNA synthetases. Genetic complementation studies and characterization of the truncated enzymes establish that the methionyl-tRNA synthetase polypeptide (676 residues) can be reduced to 547 residues without significant effect on either the activity or the stability of the enzyme. The truncated enzyme (M547) appears to be similar to a previously described fully active monomeric from of 64,000 Mr derived from the native homodimeric methionyl-tRNA synthetase (2 x 76,000 Mr) by limited trypsinolysis in vitro. According to the crystallographic three-dimensional structure at 2.5 A resolution of this trypsin-modified enzyme, the polypeptide backbone folds into two domains. The former, the N-domain, contain a crevice that is believed to bind ATP. The latter, the C-domain, has a 28 C-residue extension (520 to 547), which folds back, toward the N-domain and forms an arm linking the two domains. This study shows that upon progressive shortening of this C-terminal extension, the enzyme thermostability decreases. This observation, combined with the study of several point mutations, allows us to propose that the link made by the C-terminal arm of M547 between its N and C-terminal domains is essential to sustain an active enzyme conformation. Moreover, directing point mutations in the 528-533 region, which overhangs the putative ATP-binding site, demonstrates that this part of the C-terminal arm participates also in the specific complexation of methionyl-tRNA synthetase with its cognate tRNAs.

Acylation↗
PubMed1999

Characterisation and enzymatic properties of tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) from Pyrococcus furiosus.

The structural gene TRM1 encoding tRNA(guanine 26, N (2), N (2))-dimethyltransferase (Trm1p) of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and expressed in Escherichia coli. The corresponding recombinant enzyme (pfTrm1p) with a His6-tag at the N terminus was purified to homogeneity in three steps. The enzyme has a native molecular mass of 49 kDa (as determined by gel filtration) and is very stable to heat denaturation (t1/2at 95 degrees C is two hours). pfTrm1p is a monomer and forms a one to one complex with T7 transcripts of yeast tRNA(Phe). It methylates a single guanine residue at position 26 using S -adenosyl- l -methionine as donor of the methyl groups. Depending on the incubation temperature, the type of tRNA transcript and the ratio of enzyme to tRNA, m(2)G26 or m(2)2G26 was the main product. The addition of the second methyl group to N (2)guanine 26 takes place in vitro through a monomethylated intermediate, and the enzyme dissociates from its tRNA substrate between the two consecutive methylation reactions. Identity elements in tRNA for mono- and dimethylation reactions by the recombinant pfTrm1p were identified using in vitro T7 transcripts of 33 variants of tRNA(Asp)and tRNA(Phe)from yeast. The efficient dimethylation of G26 requires the presence of base-pairs C11.G24 and G10.C25 and a variable loop of five bases within a correct 3D-core of the tRNA molecule. These identity elements probably ensure the correct presentation of monomethylated m(2)G26 to the enzyme for the attachment of the second methyl group. In contrast, the structural requirements for monomethylation of the same guanine 26 are much more relaxed and tolerate variations in the base-pairs of the D-stem, in the size of the variable loop or distortions of the 3D-architecture of the tRNA molecule.

Cloning, Molecular↗
PubMed2004

Kinetic determinants of high-fidelity tRNA discrimination on the ribosome.

The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading. Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally. Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex. tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate.

Binding Sites↗