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M Grunberg-Manago

Publications and source records attributed to M Grunberg-Manago.

At least 91 records · Page 5Linked to original sources
PubMed1984

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.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Autogenous repression of Escherichia coli threonyl-tRNA synthetase expression in vitro.

Escherichia coli threonyl-tRNA synthetase (EC 6.1.1.3) expression has been examined in an acellular protein-synthesizing system programmed with a plasmid DNA carrying thrS, infC, pheS, and pheT, the gene for threonyl-tRNA synthetase, initiation factor 3, and the two protomers of phenylalanyl-tRNA synthetase (EC 6.1.1.20), respectively. The initial rate of synthesis of L-[35S]methionine-labeled threonyl-tRNA synthetase is markedly reduced by the addition of homogeneous RNase-free threonyl-tRNA synthetase to the assay, not by that of phenylanyl- or tyrosyl-tRNA synthetase (EC 6.1.1.1). The inhibition is 50% in the presence of 0.25 microM threonyl-tRNA synthetase and reaches 90% with 2 microM enzyme. Synthesis of mRNA in the acellular DNA-dependent protein-synthesizing system has been measured by molecular hybridization to gene-specific lambda DNA probes corresponding to thrS, pheS, and pheT. The addition to the assay of 2 microM threonyl-tRNA synthetase does not affect the extent of mRNA hybridizing to the thrS-specific DNA probe. This result is interpreted as reflecting an effect of the synthetase on its expression at the translational level. Analysis of the DNA sequence of the thrS gene predicts several potential secondary structures capable of forming in the thrS mRNA. One of these potential structures is a cloverleaf. The possible role of such structures in controlling expression of thrS is discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Sequence of the initiation factor IF2 gene: unusual protein features and homologies with elongation factors.

The gene for protein synthesis initiation factor IF2 in Escherichia coli, infB, is located downstream from nusA on the same operon. We sequenced about 3 kilobases of DNA beginning within nusA and including the entire infB structural gene plus another 392 bases downstream. This region contains no obvious strong promoter signals, but a possible transcriptional termination or pausing site occurs downstream from infB. The putative initiator codon for IF2 alpha (97,300 daltons) is AUG; that for IF2 beta (79,700 daltons) is GUG, located 471 bases downstream in the same reading frame. The codon usage for IF2 is typical of other highly expressed proteins in E. coli and suggests that IF2 mRNA is efficiently translated. IF2 alpha contains two adjacent regions (residues 104-155 and 167-214) that are rich in alanine and charged amino acids and that show striking periodicities in their sequences. These regions may alternate between flexible and helical conformations, thereby drawing together the NH2-terminal and COOH-terminal globular domains of the factor as IF2 interacts with ribosomes or tRNA. Certain regions of the DNA and protein sequences of IF2 share strong homologies with elongation factor EF-Tu and lesser homology with EF-G. In particular, a region of EF-Tu implicated in GTP binding contains sequences and secondary structure that are conserved in IF2. The homologies indicate that the genes for IF2 and the elongation factors are derived at least in part from a common ancestor.

Amino Acid Sequence↗
PubMed1983

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).

Amino Acyl-tRNA Synthetases↗
PubMed1983

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.

Alleles↗
PubMed1983

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants.

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Structural and transcriptional evidence for related thrS and infC expression.

The nucleotide sequence of thrS, the gene encoding dimeric Escherichia coli threonyl-tRNA synthetase [L-threonine:tRNAThr ligase (AMP forming), 6.1.1.3], has been determined. The structural part of the gene is found upstream of and on the same DNA coding strand as infC, the gene for translational initiation factor IF3. thrS is composed of 1,926 base pairs and accounts for a protein of molecular weight 73,906. In addition, a 336-base-pair sequence 5' to the thrS structural gene has been determined. There are only three nucleotides between the stop codon of thrS and the initiator codon of infC. The only potent transcriptional terminator structure is 55 base pairs downstream of the infC coding sequence. This implies that thrS and infC can be expressed from a polycistronic mRNA originating from a promoter upstream to thrS. Although sequence data indicate that thrS and infC are cotranscribed, in vitro transcription and RNA sequence analyses reveal the existence of a promoter within thrS. This promoter can account for the independent expression of infC as reported [Springer, M., Plumbridge, J. A., Trudel, M., Graffe, M. & Grunberg-Manago, M. (1982) Mol. Gen. Genet. 186, 247-252]. A second promoter has been located within infC and could link the expression of infC and that of the next downstream gene, pdzA. Whether these promoters function normally in vivo is an open question.

Amino Acid Sequence↗
PubMed1982

Sequence of a 1.26-kb DNA fragment containing the structural gene for E.coli initiation factor IF3: presence of an AUU initiator codon.

The nucleotide sequence of a 1.26-kb pair DNA fragment containing the structural gene for Escherichia coli initiation factor IF3 has been determined. An open reading frame of 540 nucleotides is found at the position predicted by genetic studies. The amino-acid sequence deduced from the DNA sequence accounts for a molecular weight 20 530. The important feature of the coding DNA sequence is the presence of AUU as the translational initiator codon. It is 11 bases downstream of the center of a GGAGG sequence, which can strongly pair with the sequence CCUCC near the 3' terminus of 16S rRNA. The primary DNA sequence in the region of the AUU initiator codon and its role in compensating a reduced codon-anticodon interaction in initiation complex formation are discussed.

Amino Acid Sequence↗
PubMed1982

Transcription units around the gene for E. coli translation initiation factor IF3 (infC).

The coding properties have been analyzed of in vitro constructed lambda recombinant phages carrying E. coli DNA fragments from around the structural gene for translation initiation factor IF3 (infC). This study shows that infC is expressed independently of the promoter of the threonyl-tRNA synthetase (thrS), which is the genes immediately preceding infC. It also shows that the two genes following infC, namely pheS and pheT, which form the phenylalanyl-tRNA synthetase operon, are not expressed from infC's promoter. Thus the four characterized genes of that region that were previously thought to be transcribed in the same direction are now shown to be expressed in vivo as three separate transcription units.

DNA, Bacterial↗
PubMed1982

Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli.

A novel method, not relying on genetic complementation of a mutation, was used to clone a gene for translational initiation factor IF2. Two clones from a cosmid library of total Escherichia coli DNA were isolated for their ability to overproduce IF2 in vivo as determined by quantitative immunoblotting. "Maxicell" analysis of cosmid-encoded proteins and specific immune precipitation of the labeled proteins showed that the structural gene for IF2 (inf B) had been cloned. Subcloning fragments from the original cosmids located the inf B gene to a 4.8-kilobase pair HindIII/BamHI fragment. This fragment has been inserted into an integration-deficient recombinant lambda phage that lysogenizes by homology. By mapping the point of lysogenization on the E. coli chromosome, inf B has been located at 68 min, very close to argG, nusA, rpsO, and pnp. Because the gene for initiation factor IF3 is located at 38 min on the chromosome, the genes for translational initiation factors are not grouped together.

Bacteriophage lambda↗
PubMed1982

Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro.

Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene. When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed. IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased. The results suggest that IF-3 alpha is slowly degraded to the beta form. Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis. Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate.

Bacterial Proteins↗
PubMed1981

Effect of oligonucleotide AGAGGAGGU on protein synthesis in vitro.

The oligonucleotide AGAGGAGGU, complementary to the 3' end of 16S RNA has been shown to inhibit 70S initiation complex formation on E. coli ribosomes (Taniguchi, T. and Weissmann, C., 1978. Nature, 275, 770-772). We have prepared this nonanucleotide in larger quantities by a combination of DEAE cellulose-urea chromatography and reverse phase (RPC 5) chromatography. The inhibitory effect of AGAGGAGGU on initiation complex formation has been confirmed. Furthermore, when added to a complete system for in vitro protein synthesis, the translation of Q beta RNA was inhibited by the nonanucleotide. No selectivity was observed in the inhibition of the coat protein and replicase protein synthesis. When both Q beta RNA and pAUG were present, some stimulation of pAUG binding to 70S ribosomes was observed on addition of AGAGGAGGU, as previously reported (Taniguchi and Weissmann, ibid). No effect was observed in the absence of Q beta RNA. This observation is discussed.

Escherichia coli↗
PubMed1981

[Regulation of the expression of Escherichia coli genes coding for the proteins involved in translation].

We have described our present knowledge of the regulation of the expression of genes whose products are concerned with the protein biosynthesis in E. coli. In particular, we have discussed the mechanism of translational initiation which seems to be a crucial control point. Our own work on the organisation and regulation of the expression of a group of E. coli genes coding for initiation factor IF3 and two aminoacyl-tRNA synthetases was described in detail. This work illustrates the power of the new genetic engineering technology in studies on regulation at the molecular level.

Base Sequence↗