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Methionine is the universal translation start but the first methionine is removed from most mature proteins. This review focuses on our present knowledge of the five enzymes sustaining the methionine pathway in translation initiation in Escherichia coli: methionyl-tRNA synthetase, methionyl-tRNA(fMet) formyltransferase, peptidyl-tRNA hydrolase, peptide deformylase and methionine aminopeptidase. The possible significance of retaining methionine as initiation signal is discussed.

On the basis of protein modification studies and primary structure comparison, we propose that the SKS sequence within the KMSKS signature of the class 1 aminoacyl-tRNA synthetases corresponds to the GKT(or S) sequence considered as a signature of the nucleotide triphosphate-binding site of many proteins.

Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa. A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer. Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS. Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution. In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques. In parallel, the 3-D structure has been solved at a resolution of 2.5 A. These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA f Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.

The isotopic [32P]PPi-ATP exchange activity of isoleucyl-, valyl-, histodyl-, tyrosyl-, and methionyl-tRNA synthetases from Escherichia coli are lost upon incubation in the presence of pyridoxal-5′-phosphate (PLP). When the residual activity of either isoleucyl-, valyl-, or methionyl-tRNA synthetase (monometric truncated form) was plotted as a function of the number of PLP molecules incorporated per enzyme molecule, the plots obtained appeared biphasic. Below 50% inactivation of these enzymes, PLP incorporation varied linearly with the isotopic exchange measurements, and extrapolation of the first half of the plot indicated a stoichiometry of 1.10±0.05 mol of PLP incorporated per mol of 100% inactivated synthetase. Beyond 50% inactivation, the graph deviated from its initial slope, and up to 4–5 mol of PLP were incorporated per mol of synthetase at the highest used PLP concentrations. In the cases of homodimeric histidyl- and tyrosyl-tRNA synthetases, extrapolation of the graph at 100% inactivation indicated 2.8 ± 0.1 and 2.4±0.1 mol of PLP incorporated per mol of enzyme, respectively, PLP-labeled peptides were obtained through trypsin digestion and RPLC purification, prior to Edman degradation analysis. PLP-labeled residues were identified as lysines 132, 332, 335 and 402 of monomeric methionyl-tRNA synthetase, lysines 332, 335, 402, 465, 596 and 640 of native dimeric methionyl-tRNA synthetase, lysines 22, 117, 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559, 593 and 909 of valyl-tRNA synthetase, lysines 2, 118, 369 and 370 of histidyl-tRNA synthetase and lysine 237 of tyrosyl-tRNA synthetase. In addition, the amino terminal residue of the polypeptide chain(s) of either isoleucyl-, valyl-, histidyl- or methionyl-tRNA synthetases was found labeled. Among these residues, lysines 332, 335 and 402 of monomeric methionyl-tRNA synthetase as well as lysines 332, 335, 402 and 596 of dimeric methionyl-tRNA synthetase, lysines 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559 of valyl-tRNA synthetase, lysines 2, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase were labeled in the presence of PLP concentrations smaller than or equal to 1 mM, and are shown to be critical for the activity of the enzymes. It is concluded that these residues participate to the binding sites of the phosphates of ATP on the studied synthetases.

Summary Recent affinity labeling studies have led to the identification of lysine residues at the CCA binding site of tRNA in Escherichia coli methionyl- and tyrosyl-tRNA synthetases. The comparison of the labeled peptides to the known primary structures of the aminoacyl-tRNA synthetases reveals new sequence similarities among this family of enzymes. These similarities include a ‘constant’ lysine residue whose functional significance is discussed. Moreover, a systematic computer analysis was conducted to search for similarities between the aminoacyl-tRNA synthetases taken as pairs.

Resume Utilisant differentes ribonucleases, nous avons etudie le schema de digestion des deux tRNA acceptant la methionine : l'initiateur tRNA f Met et l'elongateur tRNA m Met d' E. coli . Les positions et intensites des clivages sont comparees a celles obtenues lorsque les tRNA sont complexes a la methionyl-tRNA synthetase. Nos resultats compares a d'autres etudes, suggerent un schema general d'interaction entre les tRNA et leurs synthetases correspondantes comprenant la tige amino-acide et la region de l'anticodon. En outre, une absence d'implication de la region centrale et particulierement du bras supplementaire semble etre un trait propre au seul tRNA f Met initiateur.

Resume Des etudes recentes de Fayat et al. (Eur. J. Biochem., 78 (1977), 333–336) ont montre que la methionyl-tRNA synthetase d'Escherichia coli pouvait etre purifiee par chromatographie d'affinite sur un gel d'agarose-hexyl-adenosine-5′-phosphate. La retention de l'enzyme sur le gel conditionnee par la presence de l -methioninol qui renforce l'affinite de l'enzyme pour le nucleotide immobilise. Dans cet article, le comportement sur le gel de plusieurs autres aminoacyl-tRNA synthetases purifiees est examine en presence d'analogues de leurs acides amines prives du groupement COO−. Pour chaque enzyme, la concentration de l'analogue et celle du magnesium ont ete variees dans le tampon d'equilibration du gel. On a ainsi trouve des conditions permettant l'adsorption specifique sur le gel des isoleucyl-, valyl-, tryptophanyl- et tyrosyl-tRNA synthetases d'Escherichia coli, et des methionyl- et tyrosyl-tRNA synthetases de Bacillus stearothermophilus. Toutes ces enzymes ont pu etre eluees du gel par addition d'un exces de leur acide amine au tampon d'equilibration. Cette elution est la consequence d'un effet antagoniste de l'acide amine sur l'interaction entre l'enzyme et le nucleotide. Par contre, on n'a pas pu mettre en evidence une interaction des phenylalanyl-, leucyl-, lysl-, histidyl- et cysteinyl-tRNA synthetases d'Escherichia coli avec le gel d'agarose-hexyl-adenosine-5′-phosphate. Enfin, dans le cas de la threonyl-tRNA synthetase d'Escherichia 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 d'equilibration du gel.

Diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) alpha, beta-phosphorylase from yeast Saccharomyces cerevisiae catalyzes two reactions: Ap4A cleavage and nucleoside diphosphate--phosphate (NDP-Pi) exchange. In both reactions phosphate can be substituted by arsenate, chromate, molybdate, tungstate or vanadate. In the presence of each anion, nucleoside 5'-monophosphate (NMP) always accumulates as a product of the reaction. This indicates that an unstable NMP anion is formed as an intermediate.

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and its expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance of pheS , the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a neglegible rate.

Resume Une demonstration directe de la dissociation reversible de la forme dimerique native de la methionyl-tRNA synthetase de E. coli est apportee par diffusion de neutrons aux petits angles en utilisant la forme deuteree de l'enzyme. Les parametres structuraux de l'enzyme completement deuteree sont tres semblables a ceux de l'enzyme hydrogenee. L'analyse des variations d'intensite et de rayons de giration en fonction du contraste pour un melange stoechiometrique des deux formes de dimeres permet de caracteriser un dimere hybride forme d'un protomere hydrogene et d'un deuxieme deutere. La separation de la contribution de chacun des protomeres a la diffusion conduit a une estimation du rayon de giration du protomere in situ et a celle de la distance entre les centres de masse de chaque protomere dans le dimere.

Transformation of an E. coli strain with a recombinant plasmid DNA (pB1) encoding the genes for phenylalanyl- and threonyl-tRNA synthetases causes overproduction of these enzymes by about 100- and 5-fold, respectively. A possible effect of the overproduction of the two aminoacyl-tRNA synthetases on intracellular cognate tRNA levels has been searched for by comparing tRNAThr and tRNAPhe aminoacylation capacities in the RNA extracts from strains carrying pB1 or pBR322 plasmid DNA. The answer is that the levels of these tRNAs are not changed by selective increase of the cognate synthetases.

Summary Phenylalanyl tRNA synthetase from Escherichia coli K-12 (strain CA 244) was purified by a procedure which differs from that developed by others for the isolation of the enzyme from strains B and K-10. The purified enzyme appears homogenous on sedimentation equilibrium analysis and gel electrophoresis. Its specific activity in the aminoacylation of tRNA is identical to that of the enzyme from strain K-10. However, its molecular weight and subunit structure differ radically from those reported earlier for the enzymes from strains B and K-10. The molecular weight is 240,000 based on equilibrium sedimentation and light scattering measurements and 270,000 as deduced from Sephadex G200 gel filtration and gel electrophoresis of the subunits in the presence of sodium dodecyl sulfate. The results are consistent with a tetrameric structure of the type α 2 β 2 , composed of two kinds of subunits with molecular weights of 37,000 and 98,000, respectively.