Michèle Granger-Schnarr

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

Abstract 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.

The carboxy-terminal domain of the LexA repressor oligomerises essentially as the entire protein.

The ability of the isolated carboxy‐terminal domain of the LexA repressor of Escherichia coli to form dimers and tetramers has been investigated by equilibrium ultracentrifugation. This domain, that comprises the amino acids 85–202, is readily purified after self‐cleavage of the LexA repressor at alkaline pH. It turns out that the carboxy‐terminal domain forms dimers and tetramers essentially as the entire LexA repressor. The corresponding association constants were determined after non‐linear least squares fitting of the experimental concentration distribution. A dimer association constant of K 2 = 3 × 104 M−1 and a tetramer association constant of K 4 = 2 × 104 M−1 have been determined. Similar measurements on the entire LexA repressor [(1985) Biochemistry 24, 2812–2818] gave values of K 2 = 2.1 × 104 M−1 and K 4 = 7.7 × 104 M−1. Within experimental error the dimer formation constant of the carboxy‐terminal domain may be considered to be the same as that of the entire repressor whereas the isolated domain forms tetramers slightly less efficiently. It should be stressed that the potential error in K 4 is higher than that in K 2. The overall conclusion is that the two structural domains of LexA have also well‐defined functional roles: the amino‐terminal domain interacts with DNA and the carboxy‐terminal domain is involved in dimerisation reinforcing in this way the binding of the LexA repressor to operator DNA.

Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and western blotting

A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes. The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A. After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation. Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase. The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt. A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator. The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment.

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.

In vitro study of the interaction of the LexA represser and the UvrC protein with a uvrC regulatory region

The in vitro interaction of the LexA repressor with a regulatory region of the uvrC gene has been studied by polyacrylamide gel electrophoresis. Although the uvrC promoter region shows some homology with the canonic LexA binding site, no specific binding of the repressor to this DNA sequence could be observed, but only a cooperative nonspecific binding. By the same technique we show that the UvrC protein does not bind specifically to this regulatory DNA sequence either, although the protein is able to bind nonspecifically and cooperatively to the double‐stranded DNA fragment.

A mutant LexA repressor harboring a cleavage motif cysteine-glycine remains inducible

Using site‐directed mutagenesis of the lexA gene we have changed the amino acid Ala‐84 of the LexA repressor for a cysteine. The reason for this change was the striking homology between LexA and UmuD and the comparable size of the two amino acid side chains. Using an in vivo repression/induction assay it is shown that the LexA‐Cys‐84 mutant remains inducible by mitomycin C and UV irradiation essentially in the same way as the wild‐type repressor.

Phosphorylation of Escherichia coli proteins during the SOS response

1. The phosphorylation of Escherichia coli proteins was analyzed comparatively before and after induction of the SOS response in a temperature-sensitive mutant strain. 2. The presence of phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after labelling with radioactive orthophosphate in vivo or radioactive adenosine triphosphate in vitro. 3. Significant changes in the intensity of protein labelling were observed upon induction of the SOS functions: six proteins were found to be more phosphorylated while two others were less phosphorylated. Moreover, five additional proteins appeared to become phosphorylated exclusively during the SOS response. The molecular mass and isoelectric point of these various proteins were determined. 4. For most proteins, the changes in the pattern of protein phosphorylation were concomitant with variations in the amount of protein synthesized. 5. The changes in the pattern of phosphoproteins observed during the SOS response were not due to the temperature shift required experimentally for expressing the SOS phenotype. 6. Phosphorylation was found to be catalyzed by protein kinases that modify amino acid residues at hydroxyl groups in protein substrates. 7. Both in vivo and in vitro studies brought evidence that neither RecA nor LexA, the two key regulatory proteins of the SOS functions, were capable of undergoing phosphorylation.