Sylvain Blanquet

Crystal structure of methionyl-tRNAfMet transformylase complexed with the initiator formyl-methionyl-tRNAfMet.

The crystal structure of Escherichia coli methionyl‐tRNAfMet transformylase complexed with formyl‐methionyl‐tRNAfMet was solved at 2.8 Å resolution. The formylation reaction catalyzed by this enzyme irreversibly commits methionyl‐tRNAfMet to initiation of translation in eubacteria. In the three‐dimensional model, the methionyl‐tRNAfMet formyltransferase fills in the inside of the L‐shaped tRNA molecule on the D‐stem side. The anticodon stem and loop are away from the protein. An enzyme loop is wedged in the major groove of the acceptor helix. As a result, the C1‐A72 mismatch characteristic of the initiator tRNA is split and the 3′ arm bends inside the active centre. This recognition mechanism is markedly distinct from that of elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T‐stem side.

Crystal structure at 1.2 Å resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase

Peptidyl‐tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl‐tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl‐tRNA hydrolase could be solved at 1.2 Å resolution. It indicates a single α/β globular domain built around a twisted mixed β‐sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica. This similarity allowed the characterization by site‐directed mutagenesis of several residues of the active site of peptidyl‐tRNA hydrolase. These residues, strictly conserved among the known peptidyl‐tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C‐end of a neighbouring peptidyl‐tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl‐tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl‐tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.

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).

Structure of crystalline Escherichia coli methionyl-tRNA(f)Met formyltransferase: comparison with glycinamide ribonucleotide formyltransferase.

Formylation of the methionyl moiety esterified to the 3′ end of tRNA(f)Met is a key step in the targeting of initiator tRNA towards the translation start machinery in prokaryotes. Accordingly, the presence of methionyl‐tRNA(f)Met formyltransferase (FMT), the enzyme responsible for this formylation, is necessary for the normal growth of Escherichia coli. The present work describes the structure of crystalline E.coli FMT at 2.0 A, resolution. The protein has an N‐terminal domain containing a Rossmann fold. This domain closely resembles that of the glycinamide ribonucleotide formyltransferase (GARF), an enzyme which, like FMT, uses N‐10 formyltetrahydrofolate as formyl donor. However, FMT can be distinguished from GARF by a flexible loop inserted within its Rossmann fold. In addition, FMT possesses a C‐terminal domain with a beta‐barrel reminiscent of an OB fold. This latter domain provides a positively charged side oriented towards the active site. Biochemical evidence is presented for the involvement of these two idiosyncratic regions (the flexible loop in the N‐terminal domain, and the C‐terminal domain) in the binding of the tRNA substrate.

Antico-operative binding of initiator transfer RNAMet to methionyl-transfer RNA synthetase from Escherichia coli: Neutron scattering studies

The interaction of methionyl-tRNA synthetase with initiator tRNAMet has been investigated by neutron scattering. On the basis of parallel fluorescence measurements, two types of titrations have been performed. (1) In the presence of 10 mm-MgCl2, a condition which insures antico-operative binding of two tRNA molecules to the enzyme dimer. (2) With saturating amounts of 5′-AMP and l-methioninol, in the presence of 50 mm-MgCl2, conditions which allow two transfer RNA molecules to bind the dimer with very similar affinities. Varying the solvent density (2H2O fraction) in the samples has allowed the identification by neutron scattering of changes in the radius of gyration and in the degree of dissociation of the enzyme dimer upon tRNA binding. In buffer containing 10 mm-MgCl2, at each contrast studied, the binding process involves two steps. Firstly, one tRNAmetf molecule binds easily to one dimeric enzyme molecule with an associated decrease of the radius of gyration of the enzyme moiety. The centre of mass of this tRNA lies very close to the centre of mass of the protomer with which it associates. Then, at higher tRNA concentration, a second tRNA molecule binds to the enzyme. However, the affinity of this second site is very much weaker. With the binding of the second tRNA, the radius of gyration of the enzyme moiety increases markedly. Concomitant limited dissociation of the dimer is suggested by the experimental data. These observations combined with the fact that, in 50 mm-MgCl2 both the increased radius of gyration and the partial dissociation of the enzyme are accomplished in the absence of tRNA and remain unaffected upon binding one or two tRNA, confirm that the hindrance to binding a second tRNA in 10 mm-MgCl2 arises from the constrained conformation of the one tRNA-enzyme complex.

The C-terminal domain of peptide deformylase is disordered and dispensable for activity

Upon trypsinolysis, the 18 C‐terminal residues of Escherichia coli peptide deformylase were removed but the resulting form exhibited full activity. Moreover, a mutant fms gene encoding the first 145 out of the 168 residues of the enzyme was able to complement a fms(Ts) strain and exhibited full activity. Upon progressive truncation up to residue 139, both activity and stability decreased up to complete inactivation. Mutagenesis of residues of the 138–145 region highlights the importance of Leu‐141 and Phe‐142. N‐Terminal deletions were also carried out. Beyond two residues off, the enzyme showed a dramatic instability. Finally, NMR and thermostability studies of the full‐length enzyme and comparison to the 1–147 form strongly suggest that the dispensable residues are disordered in solution.

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 tRNAsMet from E. coli and rabbit liver, respectively, has been resolved. The statistical rate constants for the formation, kR, and for the dissociation, kD, of the 1:1 complex of these tRNAs with the dimeric enzyme have been calculated. Identical kR values of 250 μm−1 s−1 reflect similar behaviour for antico-operative binding of both tRNAsMet to native methionyl-tRNA synthetase. Advantage was taken of the difference in extent of tryptophan fluorescence-quenching induced by the bacterial and mammalian initiator tRNAsMet 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 tRNAMet 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.

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.

Neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase and of its interaction with tRNATyr

Abstract Small-angle neutron scattering studies of Escherichia coli tyrosyl-tRNA synthetase indicate that in solution this enzyme is a dimer of Mr, 91 (±6) × 103 with a radius of gyration RG of 37.8 ± 1.1 A. The increase in the scattering mass of the enzyme upon binding tRNATyr has been followed in 20 m m -imidazole · HCl (pH 7.6), 10 m m -MgCl2, 0.1 m m -EDTA, 10 m m -2-mercaptoethanol, 150 m m -KCl. A stoichiometry of one bound tRNA per dimeric enzyme molecule was found. The RG of the complex is equal to 41 ± 1 A. Titration experiments in 74% 2H2O, close to the matching point of tRNA, show an RG of 38.5 ± 1 A for the enzyme moiety in the complex. From these values, a minimum distance of 49 A between the centre of mass of the bound tRNA and that of the enzyme was calculated. In low ionic strength conditions (20 m m -imidazole-HCl (pH 7.6), 10 m m -MgCl2, 0.1 m m -EDTA, 10 m m -2-mercaptoethanol) and at limiting tRNA concentrations with respect to the enzyme, titrations of the enzyme by tRNATyr 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 Mr and RG 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).