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I Saint-Girons

Publications and source records attributed to I Saint-Girons.

At least 19 recordsLinked to original sources
PubMed1994

The Met repressor-operator complex: DNA recognition by beta-strands.

The crystal structure of the E. coli met repressor in complex with a synthetic 19-base pair oligonucleotide reveals two dimeric repressor molecules bound to adjacent sites on the DNA. The oligonucleotide contains two adjacent repeats of an 8-mer known as a met-box, which represents the consensus of the met operator sites. Each met repressor dimer is centered on a met box and interacts with the adjacent dimer through antiparallel alpha-helices, which explained the observed cooperative nature of the binding. DNA binding takes place through the insertion of a beta-ribbon into the major groove of B-form DNA, representing a novel DNA binding motif. Sequence specificity arises from direct interactions between side chains of the beta-strands and the edges of the bases in the major groove. The local DNA conformation confers additional specificity through interactions between protein and the phosphate backbone. The repressor is activated through binding of S-adenosyl methionine (SAM), the corepressor, to the face opposite to that used for DNA binding. The lack of significant conformational change upon SAM binding, together with electrostatic calculations, suggests that DNA binding enhancement occurs through long-range electrostatic interactions.

Bacterial Proteins↗
PubMed1992

Probing met repressor-operator recognition in solution.

The three-dimensional crystal structure of the Escherichia coli methionine repressor, MetJ, complexed with a DNA operator fragment is described in an accompanying article. The complex exhibits several novel features of DNA-protein interaction. DNA sequence recognition is achieved largely by hydrogen-bond contacts between the bases and amino-acid side chains located on a beta-ribbon, a mode of recognition previously hypothesized on the basis of modelling of idealized beta-strands and DNA, and mutagenesis of the Salmonella phage P22 repressors Arc and Mnt. The complex comprises a pair of MetJ repressor dimers which bind to adjacent met-box sites on the DNA, and contact each other by means of a pair of antiparallel alpha-helices. Here we assess the importance of these contacts, and also of contacts that would be made between the C-helices of the protein and DNA in a previous model of the complex, by studying mutations aimed at disrupting them. The role of the carboxy-terminal helix face in operator binding was unclear, but we demonstrate that recognition of operator sequences occurs through side chains in the beta-strand motif and that dimer-dimer interactions are required for effective repression.

Base Sequence↗
PubMed1989

Three-dimensional crystal structures of Escherichia coli met repressor with and without corepressor.

The three-dimensional crystal structure of met repressor, in the presence or absence of bound corepressor (S-adenosylmethionine), shows a dimer of intertwined monomers, which do not have the helix-turn-helix motif characteristic of other bacterial repressor and activator structures. We propose that the interaction of met repressor with DNA occurs through either a pair of symmetry-related alpha-helices or a pair of beta-strands, and suggest a model for binding of several dimers to met operator regions.

Apoproteins↗
PubMed1989

Cooperative tandem binding of met repressor of Escherichia coli.

We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.

Apoproteins↗
PubMed1988

Crystallization of the met repressor from Escherichia coli.

The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit. Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).

Amino Acid Sequence↗
PubMed1988

Methionine biosynthesis in Enterobacteriaceae: biochemical, regulatory, and evolutionary aspects.

The genes coding for the enzymes involved in methionine biosynthesis and regulation are scattered on the Escherichia coli chromosome. All of them have been cloned and most have been sequenced. From the information gathered, one can establish the existence (upstream of the structural genes coding for the biosynthetic genes and the regulatory gene) of "methionine boxes" consisting of two or more repeats of an octanucleotide sequence pattern. The comparison of these sequences allows the extraction of a consensus operator sequence. Mutations in these sequences lead to the constitutivity of the vicinal structural gene. The operator sequence is the target of a DNA-binding protein--the methionine aporepressor--which has been obtained in the pure state, for which S-adenosylmethionine acts as the corepressor. Mutations in the corresponding gene lead to the constitutive expression of all the methionine structural genes. The physicochemical properties of the methionine aporepressor are being investigated.

Base Sequence↗
PubMed1987

Fourier transform infrared investigation of the Escherichia coli methionine aporepressor.

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.

Apoproteins↗
PubMed1986

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine.

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.

Escherichia coli↗
PubMed1986

Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase.

Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme. This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis. Circular dichroism spectroscopy showed a significant change of the secondary structure. This mainly corresponds to a reduction in alpha-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%). Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin. Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin. This seems to indicate that the "loosening" of the three-dimensional structure of E. coli adenylate kinase by proline----serine substitution is largely compensated for when an enzyme X ATP or enzyme X Ap5A complex is formed.

Adenine Nucleotides↗
PubMed1986

Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region.

The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of beta-cystathionase, the MetC gene product. The metC gene (1185 nucleotides) encodes a protein having 395 amino acid residues. The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine genes that are controlled by the product of the pleiotropic regulatory metJ gene. The deduced amino acid sequence of beta-cystathionase showed extensive homology with that of the MetB protein (cystathionine gamma-synthase) that catalyzes the preceding step in methionine biosynthesis. The homology strongly suggests that the structural genes for the MetB and MetC proteins evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1985

Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.

Bacterial Proteins↗
PubMed1984

Structure and autoregulation of the metJ regulatory gene in Escherichia coli.

The nucleotide sequence of the Escherichia coli metJ regulatory gene (312 nucleotides) has been determined as well as that of two mutations located within the gene. Analysis of the sequence downstream from the metJ gene has revealed inverted repeats homologous to several intercistronic regions, also reported to occur between operons. A hybrid protein that contains the 55 first amino acid residues of the metJ protein substituting for the 8 amino acid residues at the NH2 terminus of beta-galactosidase was produced by gene fusion. The hybrid protein retaining beta-galactosidase activity was purified. Its amino-terminal sequence was determined and this allowed us to locate the translational start codon of the metJ gene. Evidence was provided for autoregulation by repression of the metJ gene. By sequencing upstream from metJ, the region situated between the metJ and metB genes was found to contain putative operator structures that we propose to call "Met boxes."

Alleles↗
PubMed1983

Structure of the metJBLF cluster in Escherichia coli K12. Sequence of the metB structural gene and of the 5'- and 3'-flanking regions of the metBL operon.

The total nucleotide sequence (1,158 nucleotides) of the metB gene of Escherichia coli coding for cystathionine gamma-synthase (386 amino acid residues, Mr = 41,503/chain) is presented. The nucleotide sequences of the flanking regions of the metB and metL genes are also presented. Analysis of these sequences and identification of a promoter region upstream from the metB gene confirms that metB and metL form an operon. The transcription direction is from metB to metL; the start site of the gene transcription has been determined. There is no structural evidence of a classical attenuation mechanism in the regulation of this operon coding for enzymes implicated in an amino acid biosynthetic pathway. Finally, the overall organization of the metJBLF gene cluster is discussed.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12.

The entire threonine operon (thrABC) of Escherichia coli K12 was cloned, and the nucleotide sequence of the thrC gene and its 3' flanking region was determined. The translation initiation codon was identified by sequencing the N-terminal part of threonine synthase, the thrC gene product. Analysis of the deduced protein sequence (428 amino acid residues) revealed a region of homology, 35 amino acids long, between the three enzymes encoded by the threonine operon. During examination of the nucleotide sequence of the 1045 base pair fragments following the thrC gene, we detected some potential rho-independent and rho-dependent transcription termination signals.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of metF, the E. coli structural gene for 5-10 methylene tetrahydrofolate reductase and of its control region.

The nucleotide sequence of the E.coli metF gene (888 nucleotides), coding for 5-10 methylene tetrahydrofolate reductase, has been determined. The metF gene product was identified in maxicells and found to be a protein of subunit molecular weight 33,000, in agreement with the size of the coding region. The starting point for metF transcription was determined by S1 nuclease mapping. No structural evidence was found for an attenuation mechanism regulating the independent metF transcriptional unit. Comparison of the regulatory region preceding the metF structural gene with the 5' flanking region of the metBL operon shows some homology spanning 24 nucleotides. These homologous sequences could be operator structures belonging to the two transcriptional units, metF and metBL, and recognized by the same regulatory protein.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗