Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius

The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius , was purified almost to homogeneity from dnrI expression strains of Escherichia coli and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified His-tagged or native DnrI was used to conduct DNA-binding assays by gel mobility-shift analysis, and the results showed no significant difference in the DNA-binding activity of native or His-tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG–dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA-binding sites for DnrI extended from upstream of the −10 to −35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG – dpsE intergenic region and about 80 bp of the dnrC – dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6–10 bp with a 5'-TCGAG-3' consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.     

Crystal structure of a gamma-butyrolactone autoregulator receptor protein in Streptomyces coelicolor A3(2)

The gamma-butyrolactone-type autoregulator/receptor systems in the Gram-positive bacterial genus Streptomyces regulate morphological differentiation or antibiotic production, or both. The autoregulator receptors act as DNA-binding proteins, and on binding their cognate ligands (gamma-butyrolactones) they are released from the DNA, thus serving as repressors. The crystal structure of CprB in Streptomyces coelicolor A3(2), a homologue of the A-factor-receptor protein, ArpA, in Streptomyces griseus, was determined. The overall structure of CprB shows that the gamma-butyrolactone receptors belong to the TetR family. CprB is composed of two domains, a DNA-binding domain and a regulatory domain. The regulatory domain contains a hydrophobic cavity, which probably serves as a ligand-binding pocket. On the basis of the crystal structure of CprB and on the analogy of the characteristics of ligand-TetR binding, the binding of gamma-butyrolactones to the regulatory domain of the receptors is supposed to induce the relocation of the DNA-binding domain through conformational changes of residues located between the ligand-binding site and the DNA-binding domain, which would result in the dissociation of the receptors from their target DNA.     

Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.     

Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.