scholarly journals Autoregulation of hpdR and its effect on CDA biosynthesis in Streptomyces coelicolor

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2641-2648 ◽  
Author(s):  
Haihua Yang ◽  
Yang An ◽  
Linqi Wang ◽  
Shuli Zhang ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Promoter Region ◽  
Streptomyces Coelicolor ◽  
Binding Activity ◽  
Catabolic Gene ◽  
Mobility Shift ◽  
S1 Nuclease ◽  
Dna Binding Activity ◽  
Electrophoretic Mobility Shift Assays ◽  
Positive Effect

HpdR, an IclR-family regulator in Streptomyces coelicolor, is a substrate-dependent repressor for the tyrosine catabolic gene hppD. In this study, S1 nuclease protection assays revealed that hpdR is subject to a negative autoregulation. Purified HpdR showed specific DNA-binding activity for the promoter region of hpdR, indicating that the autoregulation of hpdR is performed directly. The disruption of hpdR led to reduced production of CDA by S. coelicolor J1501, suggesting a positive effect of hpdR on CDA biosynthesis. Electrophoretic mobility shift assays showed that HpdR specifically bound to the promoter region of hmaS (SCO3229 in the CDA gene cluster), encoding 4-hydroxymandelic acid synthase. Disruption of hmaS in J1501 abolished CDA production. It is possible that hpdR regulates CDA biosynthesis by controlling the transcription of hmaS.

scholarly journals RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complex

1999 ◽  
Vol 19 (5) ◽  
pp. 3674-3683 ◽  
Author(s):  
Patrick C. Swanson ◽  
Stephen Desiderio
Keyword(s):  
Electrophoretic Mobility ◽  
Binding Activity ◽  
Cross Linking ◽  
Mobility Shift ◽  
Initiation Complex ◽  
Dna Binding Activity ◽  
Recombination Signal Sequences ◽  
Rag 1 ◽  
Electrophoretic Mobility Shift Assays ◽  
Single Subunit

ABSTRACT V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

scholarly journals The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10

1998 ◽  
Vol 180 (18) ◽  
pp. 4922-4928 ◽  
Author(s):  
Leonid A. Sitailo ◽  
Alexander M. Zagariya ◽  
Patrick J. Arnold ◽  
Gayatri Vedantam ◽  
David W. Hecht
Keyword(s):  
Specific Binding ◽  
Bacteroides Fragilis ◽  
Binding Activity ◽  
Inverted Repeats ◽  
Mobility Shift ◽  
E Coli ◽  
Dna Binding Activity ◽  
Footprint Analysis ◽  
Nick Site ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer inBacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriTregion. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgAwas cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognateoriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to theoriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

scholarly journals Identification of Residues Responsible for the Defective Virulence Gene Regulator Mga Produced by a Natural Mutant of Streptococcus pyogenes

2005 ◽  
Vol 187 (17) ◽  
pp. 5955-5966 ◽  
Author(s):  
Cheryl M. Vahling ◽  
Kevin S. McIver
Keyword(s):  
Streptococcus Pyogenes ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Binding Activity ◽  
Human Pathogen ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Naturally Occurring ◽  
Electrophoretic Mobility Shift Assays ◽  
Regulated Promoters

ABSTRACT Mga is a transcriptional regulator in the pathogen Streptococcus pyogenes that positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of “gain-of-function” mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen.

scholarly journals SufR, a [4Fe-4S] Cluster-Containing Transcription Factor, Represses the sufRBDCSU Operon in Streptomyces avermitilis Iron-Sulfur Cluster Assembly

2020 ◽  
Vol 86 (18) ◽  
Author(s):  
Yaqing Cheng ◽  
Mengya Lyu ◽  
Renjun Yang ◽  
Ying Wen ◽  
Yuan Song ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Binding Activity ◽  
Streptomyces Avermitilis ◽  
Assembly System ◽  
Cluster Assembly ◽  
Mobility Shift ◽  
Content Type ◽  
Dna Binding Activity ◽  
Iron Sulfur

ABSTRACT Iron-sulfur (Fe-S) clusters are ubiquitous and versatile inorganic cofactors that are crucial for many fundamental bioprocesses in nearly all organisms. How cells maintain Fe-S cluster homeostasis is not well understood in Gram-positive bacteria. Genomic analysis showed that the Suf system, which is encoded by the sufRBDCSU operon, is the sole Fe-S cluster assembly system in the genus Streptomyces. Streptomyces avermitilis is the industrial producer of avermectins, which are widely used as agricultural pesticides and antiparasitic agents. sufR (SAV6324) encodes a putative ArsR-family transcriptional regulator, which was characterized as a repressor of the sufRBDCSU operon in this investigation. Spectroscopy and mass spectrometry demonstrated that anaerobically isolated SufR contained an oxidation-sensitive [4Fe-4S] cluster and existed as a homodimer. Electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analyses revealed that [4Fe-4S]-SufR bound specifically and tightly to a 14-bp palindromic sequence (CAAC-N6-GTTG) in the promoter region of the sufR operon, repressing expression of the sufRBDCSU operon. The presence of the [4Fe-4S] cluster is critical for the DNA-binding activity of SufR. Cys182, Cys195, and Cys223 in the C-terminal region of SufR are essential for [4Fe-4S] cluster coordination, but Cys178 is not. The fourth non-Cys ligand in coordination of the [4Fe-4S] cluster for SufR remains to be identified. The findings clarify the transcriptional control of the suf operon by [4Fe-4S] SufR to satisfy the various Fe-S cluster demands. SufR senses the intracellular Fe-S cluster status and modulates the expression of the sole Fe-S cluster assembly system via its Fe-S cluster occupancy. IMPORTANCE Fe–S clusters function as cofactors of proteins controlling diverse biological processes, such as respiration, photosynthesis, nitrogen fixation, DNA replication, and gene regulation. The mechanism of how Actinobacteria regulate the expression of the sole Fe-S cluster assembly system in response to the various Fe–S cluster demands remains to be elucidated. In this study, we showed that SufR functions as a transcriptional repressor of the sole Fe-S cluster assembly system in the avermectin producer S. avermitilis. [4Fe-4S]-SufR binds to the promoter region of the suf operon and represses its expression. When Fe-S cluster levels are insufficient, SufR loses its [4Fe-4S] cluster and DNA-binding activity. Apo-SufR dissociates from the promoter region of suf operon, and the expression of the suf system is strongly increased by derepression to promote the synthesis of Fe-S clusters. The study clarifies how Streptomyces maintains its Fe-S cluster homeostasis through the activity of SufR to modulate the various Fe-S cluster demands.

scholarly journals The Myb domain of LUX ARRHYTHMO in complex with DNA: expression, purification and crystallization

2016 ◽  
Vol 72 (5) ◽  
pp. 356-361 ◽  
Author(s):  
Catarina S. Silva ◽  
Xuelei Lai ◽  
Max Nanao ◽  
Chloe Zubieta
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Mobility Shift ◽  
Data Set ◽  
Dna Binding Activity ◽  
Protein Dna Interactions ◽  
Reported Data ◽  
Electrophoretic Mobility Shift Assays ◽  
Myb Domain

LUX ARRHYTHMO (LUX) is a Myb-domain transcription factor that plays an important role in regulating the circadian clock.Luxmutations cause severe clock defects and arrhythmia in constant light and dark. In order to examine the molecular mechanisms underlying the function of LUX, the DNA-binding Myb domain was cloned, expressed and purified. The DNA-binding activity of the Myb domain was confirmed using electrophoretic mobility shift assays (EMSAs), demonstrating that the LUX Myb domain is able to bind to DNA with nanomolar affinity. In order to investigate the specificity determinants of protein–DNA interactions, the protein was co-crystallized with a 10-mer cognate DNA. Initial crystallization results for the selenomethionine-derivatized protein and data-set collection statistics are reported. Data collection was performed using theMeshAndCollectworkflow available at the ESRF.

A mammalian factor that binds telomeric TTAGGG repeats in vitro

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

scholarly journals A mammalian factor that binds telomeric TTAGGG repeats in vitro.

1992 ◽  
Vol 12 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Z Zhong ◽  
L Shiue ◽  
S Kaplan ◽  
T de Lange
Keyword(s):  
Sodium Dodecyl ◽  
Binding Activity ◽  
Repeat Sequence ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Circular Dnas ◽  
Electrophoretic Mobility Shift Assays ◽  
Ttaggg Repeat

We have identified a DNA-binding activity with specificity for the TTAGGG repeat arrays found at mammalian telomeres. This factor, called TTAGGG repeat factor (TRF), is present in nuclear extracts of human, mouse, and monkey cells. TRF from HeLa cells was characterized in detail by electrophoretic mobility shift assays. It binds double-stranded TTAGGG repeats in linear and circular DNAs. Single-stranded repeats are not recognized. The optimal site for TRF appears to contain more than six contiguous TTAGGG repeats. Tandem arrays of TAGGG, TTTAGGG, TTTTAGGG, TTGGGG, and TTAGGC repeats do not bind TRF well, indicating that TRF preferentially recognizes the telomeric repeat sequence present at mammalian chromosome ends. The apparent molecular mass of this factor, based on recovery of TRF from sodium dodecyl sulfate-polyacrylamide gels, is approximately 50 kDa. We suggest that TRF binds along the length of mammalian telomeres.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

scholarly journals Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity

2002 ◽  
Vol 22 (14) ◽  
pp. 5182-5193 ◽  
Author(s):  
Fumiko Hirose ◽  
Nobuko Ohshima ◽  
Eun-Jeong Kwon ◽  
Hideki Yoshida ◽  
Masamitsu Yamaguchi
Keyword(s):  
Dna Binding ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Mobility Shift ◽  
Interacting Protein ◽  
Reciprocal Regulation ◽  
Dna Binding Activity ◽  
Expression Of Genes ◽  
Biochemical Analyses

ABSTRACT Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5′-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

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