Jean-Francois Mayaux

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.

Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form

A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.

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.

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 operons 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.

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.

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.

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.

Role of Messenger RNA Specific Secondary Structure in the Control of Gene Expression

Prokaryotic messenger RNA can form specific secondary structures involved in transcription termination. These structures can be located within as well as at the end of an operon. In the case of several aminoacid biosynthetic operons, termination sites are found between the promoter and the structural part of the operon. Regulation of operon expression can be achieved by controlling transcription termination versus read-through at these termination sites. The control involves the coupling of RNA translation to the transcription process. The present review summarizes the evidence in favour of the same kind of control mechanism in the expression of E.coli phenylalanyl-tRNA synthetase operon.