scholarly journals Purification and Characterization of the DeoR Repressor of Bacillus subtilis

2000 ◽  
Vol 182 (7) ◽  
pp. 1916-1922 ◽  
Author(s):  
Xianmin Zeng ◽  
Hans H. Saxild ◽  
Robert L. Switzer
Keyword(s):  
Bacillus Subtilis ◽  
Dissociation Constant ◽  
Gel Filtration ◽  
Direct Repeat ◽  
Mobility Shift ◽  
Apparent Dissociation Constant ◽  
Dnase I Footprinting ◽  
Operator Dna ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

ABSTRACT Transcription of the Bacillus subtilis dra-nupC-pdpoperon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of thedra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.

scholarly journals HpdR Is a Transcriptional Activator of Sinorhizobium meliloti hpdA, Which Encodes a Herbicide-Targeted 4-Hydroxyphenylpyruvate Dioxygenase

2007 ◽  
Vol 189 (9) ◽  
pp. 3660-3664 ◽  
Author(s):  
Suvit Loprasert ◽  
Wirongrong Whangsuk ◽  
James M. Dubbs ◽  
Ratiboot Sallabhan ◽  
Kumpanart Somsongkul ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Transcriptional Activator ◽  
Direct Repeat ◽  
Dnase I ◽  
Mobility Shift ◽  
Intergenic Space ◽  
Repeat Sequences ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT Sinorhizobium meliloti hpdA, which encodes the herbicide target 4-hydroxyphenylpyruvate dioxygenase, is positively regulated by HpdR. Gel mobility shift and DNase I footprinting analyses revealed that HpdR binds to a region that spans two conserved direct-repeat sequences within the hpdR-hpdA intergenic space. HpdR-dependent hpdA transcription occurs in the presence of 4-hydroxyphenylpyruvate, tyrosine, and phenylalanine, as well as during starvation.

scholarly journals Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

2002 ◽  
Vol 184 (8) ◽  
pp. 2148-2154 ◽  
Author(s):  
Susan H. Fisher ◽  
Lewis V. Wray
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mutational Analysis ◽  
Limited Growth ◽  
Mobility Shift ◽  
Regulatory Factors ◽  
Bacillus Subtilis 168 ◽  
Dnase I Footprinting ◽  
Genes Encoding ◽  
Gel Mobility Shift

ABSTRACT Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

scholarly journals Identification and Characterization of a DeoR-Specific Operator Sequence Essential for Induction ofdra-nupC-pdp Operon Expression in Bacillus subtilis

1999 ◽  
Vol 181 (6) ◽  
pp. 1719-1727 ◽  
Author(s):  
Xianmin Zeng ◽  
Hans H. Saxild
Keyword(s):  
Bacillus Subtilis ◽  
Control Region ◽  
Regulatory Region ◽  
Direct Repeat ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Operon Expression ◽  
Specific Operator

ABSTRACT The deoR gene located just upstream thedra-nupC-pdp operon of Bacillus subtilisencodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptionallacZ fusions. It was shown that all of thecis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position −60 to position −43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3′ half of the palindrome and was located between the −10 and −35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.

Mpl Ligand Enhances the Transcription of the Cyclin D3 Gene: A Potential Role for Sp1 Transcription Factor

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4208-4221 ◽  
Author(s):  
Zhengyu Wang ◽  
Ying Zhang ◽  
Jun Lu ◽  
Shinnshin Sun ◽  
Katya Ravid
Keyword(s):  
Potential Role ◽  
Binding Activity ◽  
Cyclin D3 ◽  
Protein Phosphatase 1 ◽  
Mobility Shift ◽  
Sp1 Transcription Factor ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Shifts

Cyclin D3 plays a major role in the development of polyploidy in megakaryocytes. The expression of cyclin D3 gene and the level of cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, as indicated by Northern and Western blot analyses, and by nuclear run-on assays and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at −75 to −60 bp and at −134 to −92 bp, which display binding sites for the Sp family of transcription factors. Gel mobility shift assay and supershifts with specific antibodies indicate that Sp1 binds to these regions in the cyclin D3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activity and eliminates the induction by Mpl ligand. We find that the nonphosphorylated form of SP1 has greater affinity for the cyclin D3 promoter and that the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatment results in increased levels of Sp1 protein, which also appears as nonphosphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl ligand to activate at once several Sp1-dependent genes during megakaryopoiesis.

Mpl Ligand Enhances the Transcription of the Cyclin D3 Gene: A Potential Role for Sp1 Transcription Factor

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4208-4221 ◽  
Author(s):  
Zhengyu Wang ◽  
Ying Zhang ◽  
Jun Lu ◽  
Shinnshin Sun ◽  
Katya Ravid
Keyword(s):  
Potential Role ◽  
Binding Activity ◽  
Cyclin D3 ◽  
Protein Phosphatase 1 ◽  
Mobility Shift ◽  
Sp1 Transcription Factor ◽  
Dnase I Footprinting ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Shifts

Abstract Cyclin D3 plays a major role in the development of polyploidy in megakaryocytes. The expression of cyclin D3 gene and the level of cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, as indicated by Northern and Western blot analyses, and by nuclear run-on assays and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at −75 to −60 bp and at −134 to −92 bp, which display binding sites for the Sp family of transcription factors. Gel mobility shift assay and supershifts with specific antibodies indicate that Sp1 binds to these regions in the cyclin D3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activity and eliminates the induction by Mpl ligand. We find that the nonphosphorylated form of SP1 has greater affinity for the cyclin D3 promoter and that the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatment results in increased levels of Sp1 protein, which also appears as nonphosphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl ligand to activate at once several Sp1-dependent genes during megakaryopoiesis.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals Functional Analysis of the Bacillus subtilis Zur Regulon

2002 ◽  
Vol 184 (23) ◽  
pp. 6508-6514 ◽  
Author(s):  
Ahmed Gaballa ◽  
Tao Wang ◽  
Rick W. Ye ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Metal Ion ◽  
Dna Microarrays ◽  
Optimal Growth ◽  
Zinc Uptake ◽  
Uptake System ◽  
Mobility Shift ◽  
Transport Pathway ◽  
Dnase I Footprinting ◽  
Transporter Family

ABSTRACT The Bacillus subtilis zinc uptake repressor (Zur) regulates genes involved in zinc uptake. We have used DNA microarrays to identify genes that are derepressed in a zur mutant. In addition to members of the two previously identified Zur-regulated operons (yciC and ycdHI-yceA), we identified two other genes, yciA and yciB, as targets of Zur regulation. Electrophoretic mobility shift experiments demonstrated that all three operons are direct targets of Zur regulation. Zur binds to an ∼28-bp operator upstream of the yciA gene, as judged by DNase I footprinting, and similar operator sites are found preceding each of the previously described target operons, yciC and ycdHI-yceA. Analysis of a yciA-lacZ fusion indicates that this operon is induced under zinc starvation conditions and derepressed in the zur mutant. Phenotypic analyses suggest that the YciA, YciB, and YciC proteins may function as part of the same Zn(II) transport pathway. Mutation of yciA or yciC, singly or in combination, had little effect on growth of the wild-type strain but significantly impaired the growth of the ycdH mutant under conditions of zinc limitation. Since the YciA, YciB, and YciC proteins are not obviously related to any known transporter family, they may define a new class of metal ion uptake system. Mutant strains lacking all three identified zinc uptake systems (yciABC, ycdHI-yceA, and zosA) are dependent on micromolar levels of added zinc for optimal growth.

Positive and negative transcriptional elements of the human type IV collagenase gene

1990 ◽  
Vol 10 (12) ◽  
pp. 6524-6532
Author(s):  
S M Frisch ◽  
J H Morisaki
Keyword(s):  
Core Promoter ◽  
Type Iv Collagenase ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Element ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift ◽  
Transcriptional Elements ◽  
Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

Multiple silencer elements are involved in regulating the chicken vimentin gene

1994 ◽  
Vol 14 (2) ◽  
pp. 934-943
Author(s):  
R J Garzon ◽  
Z E Zehner
Keyword(s):  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Silencer Elements ◽  
Gel Mobility Shift ◽  
Filament Protein ◽  
Silencer Element ◽  
Vimentin Gene

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

scholarly journals In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

Microbiology ◽  
2014 ◽  
Vol 160 (2) ◽  
pp. 243-260 ◽  
Author(s):  
Öykü İrigül-Sönmez ◽  
Türkan E. Köroğlu ◽  
Büşra Öztürk ◽  
Ákos T. Kovács ◽  
Oscar P. Kuipers ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Dna Microarrays ◽  
Antibiotic Production ◽  
Transcriptional Profiling ◽  
Mobility Shift ◽  
Carbohydrate Utilization ◽  
Altered Expression ◽  
Gel Mobility Shift

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR + wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.

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