scholarly journals MarR-Like Transcriptional Regulator Involved in Detoxification of Aromatic Compounds in Sulfolobus solfataricus

2007 ◽  
Vol 189 (20) ◽  
pp. 7351-7360 ◽  
Author(s):  
Gabriella Fiorentino ◽  
Raffaele Ronca ◽  
Raffaele Cannio ◽  
Mosè Rossi ◽  
Simonetta Bartolucci
Keyword(s):  
Dna Binding ◽  
Aromatic Compounds ◽  
Sulfolobus Solfataricus ◽  
Transcriptional Regulator ◽  
Aromatic Aldehydes ◽  
Transcriptional Analysis ◽  
Regulatory Sequences ◽  
Gene Transcript ◽  
Cell Defense

ABSTRACT A DNA binding protein, BldR, was identified in the crenarchaeon Sulfolobus solfataricus as a protein 5- to 10-fold more abundant in cells grown in the presence of toxic aldehydes; it binds to regulatory sequences located upstream of an alcohol dehydrogenase gene (Sso2536). BldR is homologous to bacterial representatives of the MarR (multiple antibiotic resistance) family of transcriptional regulators that mediate response to multiple environmental stresses. Transcriptional analysis revealed that the bldR gene was transcribed in a bicistronic unit composed of the genes encoding the transcriptional regulator (Sso1352) and a putative multidrug transporter (Sso1351) upstream. By homology to bacterial counterparts, the bicistron was named the mar-like operon. The level of mar-like operon expression was found to be increased at least 10-fold in response to chemical stress by aromatic aldehydes. Under the same growth conditions, similar enhanced in vivo levels of Sso2536 gene transcript were also measured. The gene encoding BldR was expressed in E. coli, and the recombinant protein was purified to homogeneity. DNA binding assays demonstrated that the protein is indeed a transcription factor able to recognize site specifically both the Sso2536 and mar-like promoters at sites containing palindromic consensus sequences. Benzaldehyde, the substrate of ADHSs, stimulates DNA binding of BldR at both promoters. The role of BldR in the auto-activation as well as in the regulation of the Sso2536 gene, together with results of increased operon and gene expression under conditions of exposure to aromatic aldehydes, indicates a novel coordinate regulatory mechanism in cell defense against stress by aromatic compounds.

scholarly journals Ligand-Controlled Proteolysis of the Escherichia coli Transcriptional Regulator ZntR

2007 ◽  
Vol 189 (8) ◽  
pp. 3017-3025 ◽  
Author(s):  
Mihaela Pruteanu ◽  
Saskia B. Neher ◽  
Tania A. Baker
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcriptional Regulator ◽  
Zinc Homeostasis ◽  
Fine Tuning ◽  
Physical Analysis ◽  
Cellular Environment ◽  
High Zinc

ABSTRACT Proteases play a crucial role in remodeling the bacterial proteome in response to changes in cellular environment. Escherichia coli ZntR, a zinc-responsive transcriptional regulator, was identified by proteomic experiments as a likely ClpXP substrate, suggesting that protein turnover may play a role in regulation of zinc homeostasis. When intracellular zinc levels are high, ZntR activates expression of ZntA, an ATPase essential for zinc export. We find that ZntR is degraded in vivo in a manner dependent on both the ClpXP and Lon proteases. However, ZntR degradation decreases in the presence of high zinc concentrations, the level of ZntR rises, and transcription of the zntA exporter is increased. Mutagenesis experiments reveal that zinc binding does not appear to be solely responsible for the zinc-induced protection from proteolysis. Therefore, we tested whether DNA binding was important in the zinc-induced stabilization of ZntR by mutagenesis of the DNA binding helices. Replacement of a conserved arginine (R19A) in the DNA binding domain both enhances ZntR degradation and abolishes zinc-induced transcriptional activation of zntA. Biochemical and physical analysis of ZntRR19A demonstrates that it is structurally similar to, and binds zinc as well as does, the wild-type protein but is severely defective in binding DNA. Thus, we conclude that two different ligands—zinc and DNA—function together to increase ZntR stability and that ligand-controlled proteolysis of ZntR plays an important role in fine-tuning zinc homeostasis in bacteria.

scholarly journals Accurate and sensitive quantification of protein-DNA binding affinity

2018 ◽  
Vol 115 (16) ◽  
pp. E3692-E3701 ◽  
Author(s):  
Chaitanya Rastogi ◽  
H. Tomas Rube ◽  
Judith F. Kribelbauer ◽  
Justin Crocker ◽  
Ryan E. Loker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Biophysical Model ◽  
Regulatory Sequences ◽  
Binding Modes ◽  
Perfect Agreement ◽  
Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

scholarly journals The Syringate O-Demethylase Gene of Sphingobium sp. Strain SYK-6 Is Regulated by DesX, while Other Vanillate and Syringate Catabolism Genes Are Regulated by DesR

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Takuma Araki ◽  
Kenta Tanatani ◽  
Naofumi Kamimura ◽  
Yuichiro Otsuka ◽  
Muneyoshi Yamaguchi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Aromatic Compounds ◽  
Regulatory System ◽  
Value Added ◽  
Transcriptional Regulator ◽  
Inducible Promoter ◽  
Repeat Sequence ◽  
Chemical Degradation ◽  
Lignin Biodegradation ◽  
Rt Pcr

ABSTRACT Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while those of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here, we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA. Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicated that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB. The ferulate catabolism genes (ferBA), under the control of a MarR-type transcriptional regulator, FerC, are located upstream of desA. RT-PCR analyses suggested that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays revealed that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps an 18-bp inverted-repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that the compounds are effector molecules of DesX. IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and to elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (encoding a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional-regulatory systems for syringate and vanillate catabolism in SYK-6.

scholarly journals DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

1997 ◽  
Vol 17 (7) ◽  
pp. 3858-3866 ◽  
Author(s):  
G Periz ◽  
L R Keller
Keyword(s):  
Marker Gene ◽  
Gene Promoter ◽  
Transcriptional Analysis ◽  
Regulatory Sequences ◽  
Promoter Regions ◽  
Tubulin Gene ◽  
Basal Expression ◽  
Acid Shock ◽  
Sequence Elements

Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

scholarly journals ParB spreading on DNA requires cytidine triphosphate in vitro

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Adam SB Jalal ◽  
Ngat T Tran ◽  
Tung BK Le
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Genetic Information ◽  
Caulobacter Crescentus ◽  
Transcriptional Regulator ◽  
Dna Binding Protein ◽  
Living Organisms ◽  
Dna Substrate

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

scholarly journals ParB spreading on DNA requires cytidine triphosphate in vitro

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
Ngat T. Tran ◽  
Tung B. K. Le
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Genetic Information ◽  
Caulobacter Crescentus ◽  
Transcriptional Regulator ◽  
Dna Binding Protein ◽  
Living Organisms ◽  
Dna Substrate

ABSTRACTIn all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

scholarly journals The LysR-Type Transcriptional Regulator CbbR Controlling Autotrophic CO2 Fixation by Xanthobacter flavus Is an NADPH Sensor

1998 ◽  
Vol 180 (6) ◽  
pp. 1411-1417 ◽  
Author(s):  
G. van Keulen ◽  
L. Girbal ◽  
E. R. E. van den Bergh ◽  
L. Dijkhuizen ◽  
W. G. Meijer
Keyword(s):  
Dna Binding ◽  
Inorganic Compounds ◽  
Calvin Cycle ◽  
Transcriptional Regulator ◽  
Binding Assays ◽  
Autotrophic Co2 Fixation ◽  
Binding Characteristics ◽  
Xanthobacter Flavus

ABSTRACT Autotrophic growth of Xanthobacter flavus is dependent on the fixation of carbon dioxide via the Calvin cycle and on the oxidation of simple organic and inorganic compounds to provide the cell with energy. Maximal induction of the cbb andgap-pgk operons encoding enzymes of the Calvin cycle occurs in the absence of multicarbon substrates and the presence of methanol, formate, hydrogen, or thiosulfate. The LysR-type transcriptional regulator CbbR regulates the expression of the cbb andgap-pgk operons, but it is unknown to what cellular signal CbbR responds. In order to study the effects of low-molecular-weight compounds on the DNA-binding characteristics of CbbR, the protein was expressed in Escherichia coli and subsequently purified to homogeneity. CbbR of X. flavus is a dimer of 36-kDa subunits. DNA-binding assays suggested that two CbbR molecules bind to a 51-bp DNA fragment on which two inverted repeats containing the LysR motif are located. The addition of 200 μM NADPH, but not NADH, resulted in a threefold increase in DNA binding. The apparentK d NADPH of CbbR was determined to be 75 μM. By using circular permutated DNA fragments, it was shown that CbbR introduces a 64° bend in the DNA. The presence of NADPH in the DNA-bending assay resulted in a relaxation of the DNA bend by 9°. From the results of these in vitro experiments, we conclude that CbbR responds to NADPH. The in vivo regulation of the cbb andgap-pgk operons may therefore be regulated by the intracellular concentration of NADPH.

scholarly journals Temporal recruitment of CCAAT/enhancer-binding proteins to early and late adipogenic promoters in vivo

2006 ◽  
Vol 36 (1) ◽  
pp. 139-151 ◽  
Author(s):  
N Salma ◽  
H Xiao ◽  
A N Imbalzano
Keyword(s):  
Dna Binding ◽  
Chromatin Immunoprecipitation ◽  
Binding Capacity ◽  
Regulatory Sequences ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Kinetics Of

The CCAAT/enhancer-binding protein (C/EBP) family of transcriptional regulators is critically important for the activation of adipogenic genes during differentiation. The C/EBPβ and δ isoforms are rapidly induced upon adipocyte differentiation and are responsible for activating the adipogenic regulators C/EBPα and peroxisome proliferator activated receptor (PPAR)γ2, which together activate the majority of genes expressed in differentiating adipocytes. However, mitosis is required following the induction of adipogenesis, and the activation of C/EBPα and PPARγ2 gene expression is delayed until cell division is underway. Previous studies have used electromobility shift assays to suggest that this delay is due, at least in part, to a delay between the induction of C/EBPβ protein levels and the acquisition of DNA binding capacity by C/EBPβ. Here we used in vivo chromatin immunoprecipitation analysis of the C/EBPα, PPARγ2, resistin, adiponectin, and leptin promoters to examine the kinetics of C/EBP protein binding to adipogenic genes in differentiating cells. In contrast to prior studies, we determined that C/EBPβ and δ were bound to endogenous regulatory sequences controlling the expression of these genes within 1–4 h of adipogenic induction. These results indicated that C/EBPβ and δ bind not only to genes that are induced early in the adipogenic process but also to genes that are induced much later during differentiation, without a delay between induction of C/EBP protein levels and DNA binding by these proteins. We also showed that each of the genes examined undergoes a transition in vivo from early occupancy by C/EBPβ and δ to occupancy by C/EBPα at times that correlate with the induction of C/EBPα protein levels, demonstrating the generality of the transition during adipogenesis and indicating that the binding of specific C/EBP isoforms does not correlate with timing of expression from each gene. We have concluded that C/EBP family members bind to adipogenic genes in vivo in a manner that follows the induction of C/EBP protein synthesis.

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