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P Oertel-Buchheit

Publications and source records attributed to P Oertel-Buchheit.

12 recordsLinked to original sources
PubMed1999

Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-kappaB transcriptional activator.

The Drosophila transcription factor Dorsal, a member of the Rel/NF-kappaB family of proteins, plays a key role in the establishment of dorsoventral polarity in the early embryo and is also involved in the immune response. Here, we present evidence that the primary transcript of dorsal can be alternatively spliced, generating Dorsal-B, a new Rel/NF-kappaB family member. Dorsal and Dorsal-B are identical in the N-terminal region, which comprises both a DNA-binding domain and a dimerization domain. However, Dorsal-B lacks the nuclear localization signal located at the end of the Rel domain of Dorsal and is totally divergent in the C-terminal portion. Although Dorsal-B by itself is not able to induce the expression of a kappaB-controlled Luciferase reporter gene, we demonstrate that its C-terminal portion has transactivating properties. Analysis of the dorsal-B expression pattern indicates that the splicing is tissue-specific and excludes a putative role in early embryogenesis. However, dorsal-B synthesis is enhanced upon septic injury, and this challenge induces a nuclear accumulation of the protein in fat body cells suggesting that it may be involved in the immune response.

3T3 Cells↗
PubMed1998

A LexA mutant repressor with a relaxed inter-domain linker.

The LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino-terminal DNA binding domain and a carboxy-terminal dimerization domain. These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained. So far, no structure of any of the full-length repressors is available. Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu-C cleavage over a segment of at least 20 amino acids, whereas the LexA wild-type hinge region is resistant to these proteases. These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter-domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy-terminal domain and/or a fairly stable local structure of the linker region.

Amino Acid Sequence↗
PubMed1998

A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli.

Interactions between proteins affect a wide variety of biological processes, such as signal transduction and control of gene expression. In order to facilitate the study of protein-protein interactions we have developed a new method for specifically detecting the heterodimerization of two heterologous proteins in the bacterium Escherichia coli. The assay is based on the simultaneous use of protein fusions with an altered specificity and a wild-type LexA repressor DNA-binding domain. We have tested this system with two well known eukaryotic dimerization domains (the Fos and Jun leucine zippers). The two interacting proteins were, respectively, fused to a wild-type and a mutant LexA DNA-binding domain. Their hetero-association is specifically measured by the transcriptional repression of a reporter gene (lacZ) controlled by a hybrid operator containing a wild-type half-site (CTGT) and a mutated operator half-site (CCGT). The hybrid operator/lacZ construct was integrated into the chromosome of the reporter strain (SU202) to avoid possible artefacts due to variations in plasmid copy number. This method should be particularly useful in those cases where one or both partners are also able to form homodimers, since the assay described here is sensitive only to the formation of heterodimers. Furthermore, this assay gives rise to a screenable red/white phenotype on MacConkey-lactose indicator plates, allowing for a genetic study of the specificity of the interaction.

Bacterial Proteins↗
PubMed1997

DNA binding and transactivation properties of Fos variants with homodimerization capacity.

The mammalian Fos and Fos-related proteins are unable to form homodimers and to bind DNA in the absence of a second protein, like c-Jun for example. In order to study the implications of hydrophobic point mutations in the c-Fox leucine zipper on DNA binding of the entire c-Fos protein, we have constructed and purified a set of Fos mutant proteins harboring one or several isoleucine or leucine residues in the five Fos zipper a positions. We show that a single point mutation in the hydrophobic interface of the c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically to an oligonucleotide duplex harboring the TRE consensus sequence TGA(C/G)TCA. This point mutation (Thr196-->Ile) is situated in the a position of the second heptade (a2) of the Fos zipper. The introduction of additional isoleucine residues in the other a positions progressively increases the DNA binding affinity of these homodimerizing Fos zipper variants. Heterodimerization of these c-Fos variants with c-Jun reveals a complex behavior, in that the DNA binding affinity of these heterodimers does not simply increase with the number of isoleucine side chains in position a. For example, a c-Fos variant harboring a wild-type Thr in position a1 aad Ile in the four other a positions (c-Fos4I) interacts more tightly with c-Jun than a variant harboring Ile in all five a positions (c-Fos5I). The same holds true for the corresponding leucine variants, suggesting that the wild-type a1 residue of the Fox zipper (Thr162) is thermodynamically relevant for Fos-Jun heterodimer formations and DNA binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better than the wild-type zipper protein, suggesting that the driving force for Fos-Jun heterodimerization is not the simple fact that the Fos protein is unable to form homodimers. These c-Fos variants were further tested for their transactivation properties in F9 and NIH3T3 cells. At low expression levels the most efficiently homodimerizing variant (c-Fos5I) activates transcription in F9 cells about 6-fold. However part of this activation may be due to the formation of heterodimers with a member of the Jun family (like JunD for example), since a wild type c-Fos expression vector confers a 3-fold activation under these conditions. In the case of the homodimerizing c-Fos variants however, this activation is abrogated at higher expression levels due to a strong inhibition of basal transcription activity.

3T3 Cells↗
PubMed1995

Fos leucine zipper variants with increased association capacity.

The Fos wild-type leucine zipper is unable to support homodimerization. This finding is generally explained by the negative net charge of the Fos zipper leading to the electrostatic repulsion of two monomers. Using a LexA-dependent in vivo assay in Escherichia coli, we show here that additional antideterminants for Fos zipper association are the residues in position a within the Fos zipper interface. If the wild-type Fos zipper is fused to the DNA binding domain of the LexA repressor (LexA-DBD), no excess repression is observed as compared with the LexA-DBD alone, in agreement with the incapacity of the wild-type Fos zipper to promote homodimerization. If hydrophobic amino acids (Ile, Leu, Val, Phe, Met) are inserted into the five a positions of a LexA-Fos zipper fusion protein, substantial transcriptional repression is recovered showing that Fos zipper homodimerization is not only limited by the repulsion of negatively charged residues but also by the nonhydrophobic nature of the a positions. The most efficient variants (harboring Ile or Leu in the five a positions) show an about 80-fold increase in transcriptional repression as compared with the wild-type Fos zipper fusion protein. In the case of multiple identical substitutions, the overall improvement is correlated with the hydrophobicity of the inserted side chains, i.e. Ile Leu > Val > Phe > Met. However at least for Val, Phe, and Met the impact of a given residue type on the association efficiency depends strongly on the heptad, i.e. on the local environment of the a residue. This is particularly striking for the second heptad of the Fos zipper, where Val is less well tolerated than Phe and Met. Most likely the a1 residue modulates the interhelical repulsion between two glutamic acid side chains in positions g1 and e2. Most of the hydrophobic Fos zipper variants are also improved in heteroassociation with a Jun leucine zipper, such that roughly half of the additional free energy of homodimerization is imported into the heterodimer. A few candidates (including the Fos wild-type zipper) deviate from this correlation, showing considerable excess heteroassociation.

3T3 Cells↗
PubMed1993

Orientation of the LexA DNA-binding motif on operator DNA as inferred from cysteine-mediated phenyl azide crosslinking.

To address the question how the recognition helix of the LexA repressor is positioned within the major groove of operator DNA we have applied a site-specific photocrosslinking approach using a LexA mutant repressor (LexA-C52) that harbors a single cysteine side chain in position 52, close to the COOH terminus of helix 3. The LexA-C52 mutant repressor has been purified and modified site-specifically with the photoreactive azido compound 4-azidophenacyl bromide, giving rise to LexA-C52*. Here we show that LexA-C52* may be selectively photocrosslinked with two adjacent bases within each operator half-site. The crosslinked bases are located, respectively, 10 and 11 base pairs from the dyad axis of the operator. The crosslinking data imply that the LexA recognition helix is oriented opposite to what is generally observed for helix-turn-helix proteins and that this helix should form a steeper angle with respect to the plane of the base pairs than is observed for standard helix-turn-helix proteins.

Azides↗
PubMed1993

Spacing requirements between LexA operator half-sites can be relaxed by fusing the LexA DNA binding domain with some alternative dimerization domains.

The dimerization domain of the LexA repressor has been replaced by two heterologous dimerization motifs: the "leucine zipper" from the jun oncogene product and the carboxy-terminal oligomerization domain of Escherichia coli lac repressor. The corresponding hybrid proteins LexA1-87-Jun zipper and LexA1-87-lac repressor have been purified and their DNA binding properties have been studied using gel mobility shift assays. Both fusion proteins form stable specific complexes with a short DNA duplex harboring the CTGT(at)4ACAG consensus sequence of the LexA repressor. This conserved DNA binding capacity distinguishes these two fusion proteins from many others containing a LexA DNA binding domain fused to different heterologous transactivation and/or dimerization domains. However the fusion proteins LexA1-87-Jun zipper and LexA1-87-lac repressor behave differently from native LexA repressor in that these fusion proteins tolerate the insertion of additional base-pairs between the two invertedly repeated CTGT motifs. LexA1-87-Jun zipper requires two CTGT motifs and tolerates the insertion of at least two additional base-pairs between these motifs, whereas LexA1-87-lac repressor requires in fact only a single CTGT motif for the formation of a specific complex detectable in gel mobility shift assays. The inability of the normal LexA repressor to form well-defined complexes with operators containing additional base-pairs in the center suggests that the LexA "hinge region" between the amino-terminal DNA binding and the carboxy-terminal dimerization domain might not be entirely flexible. In an attempt to remove a hypothetical interaction between the LexA cleavage site (which is situated within the hinge region) and the catalytic cleavage center (situated within the carboxy-terminal domain) a LexA mutant repressor containing five simultaneous mutations in the hinge region has been constructed and purified. Surprisingly this mutant repressor failed to form stable complexes detectable by the gel mobility shift assay even with the normal consensus sequence, suggesting that the LexA hinge region is more than a simple connector between the two structural domains and that its chemical nature is important not only for LexA cleavage, but also for the formation of stable LexA-DNA complexes.

Avian Proteins↗
PubMed1992

Isolation and characterization of LexA mutant repressors with enhanced DNA binding affinity.

The LexA repressor from Escherichia coli is a sequence-specific DNA binding protein that shows no pronounced sequence homology with any of the known structural motifs involved in DNA binding. Since little is known about how this protein interacts with DNA, we have selected and characterized a great number of intragenic, second-site mutations which restored at least partially the activity of LexA mutant repressors deficient in DNA binding. In 47 cases, the suppressor effect of these mutations was due to an Ind- phenotype leading presumably to a stabilization of the mutant protein. With one exception, these second-site mutations are all found in a small cluster (amino acid residues 80 to 85) including the LexA cleavage site between amino acid residues 84 and 85 and include both already known Ind- mutations as well as new variants like GN80, GS80, VL82 and AV84. The remaining 26 independently isolated second-site suppressor mutations all mapped within the amino-terminal DNA binding domain of LexA, at positions 22 (situated in the turn between helix 1 and helix 2) and positions 57, 59, 62, 71 and 73. These latter amino acid residues are all found beyond helix 3, in a region where we have previously identified a cluster of LexA (Def) mutant repressors. In several cases the parental LexA (Def) mutation has been removed by subcloning or site-directed mutagenesis. With one exception, these LexA variants show tighter in vivo repression than the LexA wild-type repressor. The most strongly improved variant (LexA EK71, i.e. Glu71----Lys) that shows an about threefold increased repression rate in vivo, was purified and its binding to a short consensus operator DNA fragment studied using a modified nitrocellulose filter binding assay. As expected from the in vivo data, LexA EK71 interacts more tightly with both operator and (more dramatically) with non-operator DNA. A determination of the equilibrium association constants of LexA EK71 and LexA wild-type as a function of monovalent salt concentration suggests that LexA EK71 might form an additional ionic interaction with operator DNA as compared to the LexA wild-type repressor. A comparison of the binding of LexA to a non-operator DNA fragment further shows that LexA interacts with the consensus operator very selectively with a specificity factor of Ks/Kns of 1.4 x 10(6) under near-physiological salt conditions.

Amino Acid Sequence↗
PubMed1991

Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study.

An increasing number of eukaryotic transcription factors interacting specifically with DNA comprise a dimerization motif called the "leucine zipper". These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif. The leucine zipper of the oncoprotein Jun is particular in that Jun may form homodimers as well as heterodimers with the oncoprotein Fos, which are however more stable than the Jun-Jun homodimers. Leucine zipper dimerization is thought to occur through a coiled-coil arrangement of parallel alpha-helices, but the rules governing the specificity of homo- and/or heterodimerization are still largely unknown. To address this question in the case of the Jun leucine zipper, we constructed a fusion protein containing the amino-terminal DNA binding domain of the LexA repressor from Escherichia coli fused to the Jun leucine zipper. This hybrid protein (LexA-JunZip) is stable in E. coli and confers much tighter repression in vivo than the DNA binding domain of LexA alone. DNA binding competition experiments with synthetic Jun and Fos leucine zipper peptides in vitro showed that the leucine zipper mediated dimerization of LexA-JunZip is essential for DNA binding of the fusion protein. The purified LexA-JunZip protein dimerizes in vitro with a dimerization constant of 2 x 10(7) M-1 at 5 degrees C. Dimerization is very sensitive to temperature, since the dimerization constant drops at 20 degrees C to 2 x 10(6) M-1 and at 30 degrees C to only 3 x 10(5) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

DNA binding properties of the LexA repressor.

The LexA repressor from Escherichia coli negatively regulates the transcription of about 20 different genes upon binding with variable affinity to single-, double- or even triple-operators as in the case of the recN gene. Binding of LexA to multiple operators is cooperative if the spacing between these operators is favorable. LexA recognizes DNA via its amino-terminal domain. The three-dimensional structure of this domain has been determined by NMR measurements. It contains three alpha-helices spanning residues 8-20, 28-35 and 41-54. In view of this structure, but also according to homology considerations and the unusual contact pattern with the DNA backbone, the LexA repressor is not a normal helix-turn-helix DNA binding protein like for example phage lambda repressor. LexA is at best a distant relative of this class of transcription factors and should probably be considered as a protein that contains a new DNA binding motif. A cluster of LexA mutant repressors deficient in DNA binding falling into the third helix (residues 41-54 bp) suggests that this helix is involved in DNA recognition.

Amino Acid Sequence↗
PubMed1990

Genetic analysis of the LexA repressor: isolation and characterization of LexA(Def) mutant proteins.

We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three alpha helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.

Amino Acid Sequence↗