Guy Fayat

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNAfMet formyltransferase☆

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA(Met) containing the acceptor stem of tRNA(fMet) and the remaining of the structure of tRNA(mMet). In addition, more than 30 variants of the genes encoding tRNA(mMet) or tRNA(fMet) have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA(mMet) became formylatable by the only change of the G1.C72 base-pair into C1-A72. It was possible to render tRNA(mMet) as good a substrate as tRNA(fMet) for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2.C71, C3.G70 and G4.C69 are positive determinants for the specific processing of methionyl-tRNA(fMet) by the formylase while the occurrence of a G.C or C.G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA(fMet).

Neutron-scattering studies of the binding of initiator tRNAMet to Escherichia coli trypsin-modified methionyl-tRNA synthetase

The trypsin-modified methionyl-tRNA synthetase, a monomer of molecular weight 64 × 103, binds one molecule of tRNAMetf with strong affinity. A separation of about 25is observed between the centres of mass of the protein and the tRNA in the 1 : 1 complex. This can be reconciled with data on the contact regions only if a conformational change is assumed for the tRNA upon binding, probably involving the CCA end. The protein moiety of the complex might be slightly more contracted than in the free conformation, but this effect is not clearly outside experimental error. There is no apparent change in the protein structure as a function of MgCl2 concentration or of added small ligands. In conditions of 10 m m -MgCl2 and limiting tRNA, a 2 : 1, enzyme: tRNA complex is formed, the second enzyme binding with considerably lower affinity. The aggregate dissociates in favour of the 1:1 complex upon increasing tRNA concentration. The results are discussed in the context of the mode of action of the dimeric native enzyme.

Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase

Comparison of the amino‐acid sequences of several methionyl‐tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in theE. coli enzyme sequence, was assayed by the use of site‐directed mutagenesis. Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino‐acid region is located at or close to the amino‐acid binding pocket of methionyl‐tRNA synthetase.

Methionyl-tRNA synthetase from Bacillus stearothermophilus : structural and functional identities with the Escherichia coli enzyme

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.

Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity.

Escherichia coli strains with abnormally high concentrations of bis(5-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.

Affinity chromatography of aminoacyl-tRNA synthetases on agarose-hexyl-adenosine-5′-phosphate

Resume Des etudes recentes de Fayat et al. (Eur. J. Biochem., 78 (1977), 333–336) ont montre que la methionyl-tRNA synthetase dEscherichia coli pouvait etre purifiee par chromatographie daffinite sur un gel dagarose-hexyl-adenosine-5′-phosphate. La retention de lenzyme sur le gel conditionnee par la presence de l -methioninol qui renforce laffinite de lenzyme pour le nucleotide immobilise. Dans cet article, le comportement sur le gel de plusieurs autres aminoacyl-tRNA synthetases purifiees est examine en presence danalogues de leurs acides amines prives du groupement COO−. Pour chaque enzyme, la concentration de lanalogue et celle du magnesium ont ete variees dans le tampon dequilibration du gel. On a ainsi trouve des conditions permettant ladsorption specifique sur le gel des isoleucyl-, valyl-, tryptophanyl- et tyrosyl-tRNA synthetases dEscherichia coli, et des methionyl- et tyrosyl-tRNA synthetases de Bacillus stearothermophilus. Toutes ces enzymes ont pu etre eluees du gel par addition dun exces de leur acide amine au tampon dequilibration. Cette elution est la consequence dun effet antagoniste de lacide amine sur linteraction entre lenzyme et le nucleotide. Par contre, on na pas pu mettre en evidence une interaction des phenylalanyl-, leucyl-, lysl-, histidyl- et cysteinyl-tRNA synthetases dEscherichia coli avec le gel dagarose-hexyl-adenosine-5′-phosphate. Enfin, dans le cas de la threonyl-tRNA synthetase dEscherichia coli, une retention sur le gel a pu etre obtenue. Cependant cette retention apparait non-specifique. Elle depend aussi bien de la presence de 1-amino-2-propanol, analogue de la threonine, que de celle de phenylalaninol ou de methioninol dans le tampon dequilibration du gel.

Binding of the anticodon domain of tRNAfMet to Escherichia coli methionyl-tRNA synthetase☆

A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro. This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2). The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence. Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).