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 Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Frank Herrmann ◽  
Manuela Hessmann ◽  
Sabine Schaertl ◽  
Karola Berg-Rosseburg ◽  
Christopher J Brown ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Trinucleotide Repeat ◽  
Radioligand Binding ◽  
Ex Vivo ◽  
Binding Assays ◽  
Pharmacological Characterization ◽  
Binding Characteristics

AbstractHuntington’s disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer’s disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer’s disease patients.

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 Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

2006 ◽  
Vol 188 (6) ◽  
pp. 2081-2095 ◽  
Author(s):  
Leah S. Feldman-Cohen ◽  
Yongping Shao ◽  
Derrick Meinhold ◽  
Charmi Miller ◽  
Wilfredo Colón ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Motif ◽  
Affinity Binding ◽  
Flanking Sequence ◽  
Binding Assays ◽  
High Affinity Binding ◽  
High Affinity

ABSTRACT Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd , ∼1 to 4 nM) and nonspecific (Kd , ∼4 μM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and λ attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the λ attR Fis site, and the role of R89 was dramatically altered by the λ attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

scholarly journals Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of Xanthobacter flavus

2003 ◽  
Vol 185 (4) ◽  
pp. 1245-1252 ◽  
Author(s):  
Geertje van Keulen ◽  
Anja N. J. A. Ridder ◽  
Lubbert Dijkhuizen ◽  
Wim G. Meijer
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
Calvin Cycle ◽  
Transcriptional Regulator ◽  
Site Directed Mutagenesis ◽  
Main Function ◽  
Xanthobacter Flavus ◽  
Negative Effect

ABSTRACT The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO2 fixation. The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap. Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional. Inverted repeat IR1 is a high-affinity CbbR binding site. The main function of this repeat is to recruit CbbR to the cbb operon promoter. In addition, it is required for negative autoregulation of cbbR expression. IR3 represents the main low-affinity binding site of CbbR. Binding to IR3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR1 also prevent binding to the low-affinity site. Although mutations in IR3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity. This is most likely due to steric hindrance of RNA polymerase by CbbR since IR3 partially overlaps with the −35 region of the cbb operon promoter. Mutations in IR2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter. This IR2 binding site is therefore critical for transcriptional activation by CbbR.

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 MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1.

1994 ◽  
Vol 14 (4) ◽  
pp. 2534-2544 ◽  
Author(s):  
L Bruhn ◽  
G F Sprague
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Specific Gene ◽  
Response Factor ◽  
Binding Assays ◽  
Yeast Saccharomyces Cerevisiae ◽  
Coregulatory Proteins ◽  
Serum Response

Complexes formed between MCM1 and several coregulatory proteins--alpha 1, alpha 2, and STE12--serve to govern transcription of the a- and alpha-specific gene sets in the yeast Saccharomyces cerevisiae. The N-terminal third of MCM1, MCM1(1-98), which includes a segment homologous to mammalian serum response factor, is capable of performing all of the functions necessary for cell-type-specific gene regulation, including DNA binding and interaction with coregulatory proteins. To explore the mechanisms by which MCM1(1-98) functions, we isolated point mutants that are specifically deficient in alpha-specific gene expression in vivo, anticipating that many of the mutants would be impaired for interaction with alpha 1. Indeed, in vitro DNA binding assays revealed that a substantial number of the mutants were specifically defective in the ability to bind cooperatively with alpha 1. Two other mutant classes were also found. One class, exemplified most clearly by substitutions at residues 22 and 27, exhibited a general defect in DNA binding. The second class, exemplified by substitutions at residues 33 and 41, was proficient at DNA binding and interaction with alpha 1 in vitro, suggesting that these mutants may be defective in achieving an alpha 1-mediated conformational change required for transcription activation in vivo. Most of the mutants defective for interaction with alpha 1 had substitutions within residues 69 to 81, which correspond to a region of serum response factor important for interaction with its coregulatory proteins. A subset of the mutants with changes in this region were also defective in the ability to bind with STE12 to DNA from an a-specific gene, suggesting that a common region of MCM1(1-98) mediates interaction with both alpha 1 and STE12. This region of MCM1 does not seem to constitute an independent domain of the protein, however, because some substitutions within this region affected DNA binding. Only two of the MCM1(1-98) point mutants showed significant defects in the ability to form complexes with alpha 2, suggesting that the mechanism by which MCM1 interacts with alpha 2 is distinct from that by which it interacts with alpha 1 and STE12.

scholarly journals Molecular mechanisms of Evening Complex activity inArabidopsis

2020 ◽  
Vol 117 (12) ◽  
pp. 6901-6909 ◽  
Author(s):  
Catarina S. Silva ◽  
Aditya Nayak ◽  
Xuelei Lai ◽  
Stephanie Hutin ◽  
Véronique Hugouvieux ◽  
...  
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Complex Activity ◽  
High Affinity ◽  
Clock Gene Expression ◽  
Repressor Complex

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX–ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

scholarly journals Genome-wide binding analyses of HOXB1 revealed a novel DNA binding motif associated with gene repression

2020 ◽  
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Es Cells ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in ES cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1 and MEIS can bind to this motif. In vivo, this motif is sufficient to direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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