Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo.

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.

Dual level control of the Escherichia coli pheST-himA operon expression. tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli have established that the pheST operon transcription is controlled by a Phe-tRNA(Phe)-mediated attenuation mechanism. More recently, the himA gene, encoding the alpha-subunit of integration host factor, was recognized immediately downstream from pheT, possibly forming part of the same transcriptional unit. By using the in-vitro transcription and S1 mapping techniques, transcription termination after pheT could be excluded, indicating that himA can be expressed from polycistronic messenger RNAs encompassing the pheST region. However, the presence of a secondary promoter able to express himA and located within pheT is demonstrated. To further investigate the regulation of the pheST-himA operon expression, genetic fusions between various parts of this operon and the lacZ gene were constructed and studied. Our results confirm the autoregulation of himA previously described, and demonstrate that it occurs through the modulation of the secondary promoter activity within pheT. Surprisingly, it is found that the pheST promoter is also submitted to the same control. Consistent with this, DNA sequences homologous to the integration host factor binding site consensus are present at the level of both promoters. However, evidence in favor of two different repressor complexes is provided. Previously observed SOS induction of the himA expression is shown to occur through the modulation of both promoter activities. Contrasting with the other genes under SOS control, the LexA protein binding site consensus sequence could not be found in the two promoter regions. This suggests that either the LexA protein directly participates in the formation of an active holorepressor, or that the product of an SOS gene is able to inhibit the formation or the binding of such a repressor. Finally, our results indicate that the pheST-himA operon expression is controlled by two different mechanisms acting independently. (1) The phenylalanyl-tRNA synthetase and the himA product expressions are controlled by an operator-repressor type mechanism, in which the himA product and the SOS network are involved. (2) Through its partial cotranscription with pheST, himA expression is also under attenuation control. The latter control may provide a way to couple the intracellular concentration of the himA product to the functional state of the translational apparatus.

Binding of the anticodon domain of tRNA(fMet) 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).

Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase.

To be aminoacylated by Escherichia coli methionyl-tRNA synthetase, a tRNA requires the presence of the methionine anticodon (CAU sequence). However, the importance in this reaction of the other nucleotides of tRNAs(Met) has still to be described. In this work, through the study of more than 35 variants of tRNAs(Met), it is shown, firstly, that the parameters of the aminoacylation reaction remain independent of the mutations affecting either the sequences or the sizes of the D-loop, D-stem and variable loop. This conclusion is illustrated by the construction and study of a tRNAf(MetCAU) with the D-stem, D-loop and very long variable loop of a class II tRNA. The resulting chimaeric tRNA is methionylated as efficiently as tRNAf(MetCAU) or tRNAm(MetCAU). Secondly, mutations affecting base 73 and base pairs 2.71 and 3.70 in the acceptor stem of tRNAf(MetCAU), as well as bases 32, 33 and 37, adjacent to the anticodon, cause a strong reduction of the rate of the aminoacylation reaction. Thirdly, it is shown that, provided it is given the acceptor stem of tRNAm(MetCAU) or tRNAf(MetCAU), a tRNA having the anticodon loop of tRNA(Met) can be converted into a substrate for methionyl-tRNA synthetase as efficient as tRNAf(MetCAU) or tRNAm(MetCAU). Finally, it is proposed that, beyond the binding of the anticodon loop to the synthetase, the sequence of the acceptor stem may strongly influence the rate of the catalytic step of the aminoacylation reaction by properly orientating the 3'-end of the tRNA towards the catalytic centre.

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.

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.

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.

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) 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).

Attenuation control of the Escherichia coli phenylalanyl-tRNA synthetase operon.

The pheST operon codes for the two subunits of the essential enzyme phenylalanyl-tRNA synthetase. The nucleotide sequence of the regulatory regions of the operon, in vitro transcription data and in vivo experiments indicate that the operon is controlled by attenuation in a way similar to many amino acid biosynthetic operons. In this work the control of the pheST operon was studied in vivo by measuring the effect of deletions in the regulatory regions on downstream expression. The presence of a strong promoter followed by an approximately 90% efficient terminator in front of the structural parts of the operon is demonstrated. An open reading frame coding for a 14 amino acid long leader peptide containing five phenylalanine residues is located between the promoter and the terminator. The presence of the transcription terminator is shown to be essential to the operon's regulation. The localization of the promoter and the terminator agrees with the results of previous in vitro experiments. It is also shown that about 30% of the transcripts covering the pheST operon come from the upstream gene, rplT, which codes for the ribosomal protein L20. Although cotranscription exists between rplT and pheST, these genes are not systematically coregulated since reducing the translation of rplT about tenfold, does not change pheST expression. The pheST operon is also shown to be derepressed by a cellular excess of phenylalanyl-tRNA synthetase. This derepression is shown to be due to the pheST attenuator.

Escherichia coli phenylalanyl-tRNA synthetase operon region. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20.

The nucleotide sequences of pheS and of the beginning of pheT have been determined. The genes pheS and pheT code, respectively, for the small and large subunits of phenylalanyl-tRNA synthetase, an alpha 2 beta 2 enzyme. Upstream from pheS the sequence shows another open reading frame of 354 nucleotides (rplT), which accounts for a protein of Mr 13,400. The product of this gene, previously named "P12", is identified as the ribosomal protein L20. The promoter for the pheS, T operon was located 368 nucleotides in front of pheS by transcription experiments in vitro. The promoter site is followed by a short open reading frame, which codes for a 14-residue peptide containing five phenylalanine residues. Immediately downstream from the stop codon of this open reading frame, the DNA sequence indicates that the transcript can be folded into three alternative secondary structures, one of which is a site of transcription termination. In vitro, 90% of transcription products initiated at the pheS, T promoter terminate at this site. However, long run-off transcripts proceeding through the terminator and covering the pheS structural gene are observed. No other transcription initiation could be detected between the terminator and the pheS structural gene. All these results are consistent with a mechanism by which phenylalanine-mediated attenuation controls the expression of phenylalanyl-tRNA synthetase. Further evidence is provided for this model by the features of pheS, T regulation in vivo (see the accompanying paper).

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro.

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.

Escherichia coli phenylalanyl-tRNA synthetase operon is controlled by attenuation in vivo.

The two subunits of phenylalanyl-tRNA synthetase are made from two adjacent, cotranscribed genes that constitute the pheS,T operon. Three different fusions between pheS,T and lac genes were constructed in order to study the regulation of the pheS,T operon in vivo. We show, using these fusions, that phenylalanyl-tRNA synthetase transcription is derepressed when the level of aminoacylated tRNAPhe is lowered by mutational alteration of the synthetase. The pheS,T operon is also derepressed in strains carrying a trpX mutation. The gene trpX codes for an enzyme that modifies both tRNATrp and tRNAPhe and a mutation in that gene causes derepression of the trp and pheA operons, both of which are controlled by attenuation. The in vivo features of the regulation of pheS,T expression described here in correlation with the DNA sequence and in vitro transcription results described in the accompanying paper by Fayat et al. indicate that phenylalanyl-tRNA synthetase is controlled by attenuation in a way analogous to several amino acid biosynthetic operons.

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in vivo.

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

IS4 transposition in the attenuator region of the Escherichia coli pheS,T operon.

A cis-acting mutation which lowers phenylalanyl-tRNA synthetase operon (pheS,T) transcription about tenfold was previously isolated on a multicopy plasmid [Plumbridge and Springer, J. Bacteriol. 152 (1982) 650-668]. This mutation has now been characterized as an IS4 element inserted in orientation II in the terminator stem of the pheS,T attenuator. The identification of the insertion as IS4 is based on (i) the nature and location of restriction sites internal to the insertion element, and (ii) the DNA sequence of both the left and right Escherichia coli::IS4 junctions. The effect of the IS4 transposition on the expression of pheS,T was studied using pheS,T::lac fusions cloned in lambda phages. IS4 integration into the leader region of the pheS,T operon was shown to abolish the miaA (trpX) allele dependence which characterizes the attenuation mechanism regulating pheS,T expression [Fayat et al., J. Mol. Biol. 171 (1983) 239-261; Springer et al., J. Mol. Biol. 171 (1983) 263-279]. The IS4 insertion site described here is compared to the other known sites and the effect of IS4 transposition on the expression of neighbouring genes is discussed.

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 the E. 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.

Effect of the overproduction of phenylalanyl- and threonyl-tRNA synthetases on tRNAPhe and tRNAThr concentrations in E. coli cells.

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.

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli.

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it 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 to 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 negligible rate.

Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase--beta-galactosidase chimeric protein.

The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

Methionyl-tRNA synthetase from E. coli--a review.

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.