scholarly journals Determination and Dissection of DNA-Binding Specificity for the Thermus thermophilus HB8 Transcriptional Regulator TTHB099

2020 ◽  
Vol 21 (21) ◽  
pp. 7929
Author(s):  
Kristi Moncja ◽  
Michael W. Van Dyke
Keyword(s):  
Dna Binding ◽  
Thermus Thermophilus ◽  
Receptor Protein ◽  
Binding Motif ◽  
Thermus Thermophilus Hb8 ◽  
E Coli ◽  
Camp Receptor ◽  
Related Proteins ◽  
Dual Regulator

Transcription factors (TFs) have been extensively researched in certain well-studied organisms, but far less so in others. Following the whole-genome sequencing of a new organism, TFs are typically identified through their homology with related proteins in other organisms. However, recent findings demonstrate that structurally similar TFs from distantly related bacteria are not usually evolutionary orthologs. Here we explore TTHB099, a cAMP receptor protein (CRP)-family TF from the extremophile Thermus thermophilus HB8. Using the in vitro iterative selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), we identified the preferred DNA-binding motif for TTHB099, 5′–TGT(A/g)NBSYRSVN(T/c)ACA–3′, and mapped potential binding sites and regulated genes within the T. thermophilus HB8 genome. Comparisons with expression profile data in TTHB099-deficient and wild type strains suggested that, unlike E. coli CRP (CRPEc), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRPEc.

scholarly journals LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2650-2660 ◽  
Author(s):  
Hideaki Takano ◽  
Yoshihiro Agari ◽  
Kenta Hagiwara ◽  
Ren Watanabe ◽  
Ryuta Yamazaki ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Regulatory Protein ◽  
Carotenoid Biosynthesis ◽  
Thermophilic Bacterium ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
S1 Nuclease ◽  
Positive Regulator ◽  
Camp Receptor

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

scholarly journals Discovering the DNA-Binding Consensus of the Thermus thermophilus HB8 Transcriptional Regulator TTHA1359

2021 ◽  
Vol 22 (18) ◽  
pp. 10042
Author(s):  
Josiah L. Teague ◽  
John K. Barrows ◽  
Cynthia A. Baafi ◽  
Michael W. Van Dyke
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Transcription Initiation ◽  
Motif Discovery ◽  
Thermus Thermophilus ◽  
Model Organism ◽  
Reverse Genetic ◽  
Thermus Thermophilus Hb8

Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5′–AWTGTRA(N)6TYACAWT–3′, which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

scholarly journals GntP Is the Escherichia coli Fructuronic Acid Transporter and Belongs to the UxuR Regulon

2004 ◽  
Vol 186 (22) ◽  
pp. 7690-7696 ◽  
Author(s):  
Cristina Bates Utz ◽  
Ann B. Nguyen ◽  
Darren J. Smalley ◽  
April B. Anderson ◽  
Tyrrell Conway
Keyword(s):  
Escherichia Coli ◽  
Protein A ◽  
Physiological Role ◽  
Major Facilitator Superfamily ◽  
Receptor Protein ◽  
E Coli ◽  
The Subject ◽  
Camp Receptor ◽  
Major Facilitator

ABSTRACT Escherichia coli has four gluconate transporters, GntP, GntU, GntT, and IdnT, which are members of the major facilitator superfamily. The physiological function of GntP was previously unknown and is the subject of this study. GntP is not induced by gluconate, and despite being located adjacent to genes involved in glucuronate catabolism, gntP does not encode a glucuronate transporter. Here we identify gntP as the gene which encodes the fructuronate transporter. We show that gntP is induced by fructuronate and is a new member of the UxuR regulon: gntP is derepressed in an uxuR strain, UxuR binds in vitro specifically to an operator site that overlaps the gntP promoter, and UxuR binding is eliminated by fructuronate. Transcription of gntP requires activation by cyclic AMP (cAMP)-cAMP receptor protein. A gntP mutant cannot grow on fructuronate but grows normally on glucuronate and gluconate. Thus, the UxuR regulon is a module of sugar acid catabolism whose physiological role is for growth on fructuronate. Glucuronate, because it proceeds through a fructuronate intermediate, must induce the UxuR regulon and must also induce the ExuR regulon, which encodes the glucuronate transporter, ExuT, and the first step in its catabolism, UxaC. Thus, hexuronate catabolism in E. coli requires both the ExuR and UxuR regulons, while fructuronate catabolism requires only the UxuR regulon.

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 Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

2004 ◽  
Vol 24 (5) ◽  
pp. 2091-2102 ◽  
Author(s):  
Chao Wei ◽  
Carolyn M. Price
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Site ◽  
Domain Architecture ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Telomere Protein ◽  
Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

Preventive action of thioethers towards in vitro DNA binding and mutagenesis in E. coli K12 by alkylating agents

1990 ◽  
Vol 245 (2) ◽  
pp. 67-74 ◽  
Author(s):  
E.Dinant Kroese ◽  
Marco J. Zeilmaker ◽  
Georges R. Mohn ◽  
John H.N. Meerman
Keyword(s):  
Dna Binding ◽  
Alkylating Agents ◽  
Preventive Action ◽  
E Coli

scholarly journals The Bacterial DNA Binding Protein MatP Involved in Linking the Nucleoid Terminal Domain to the Divisome at Midcell Interacts with Lipid Membranes

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Begoña Monterroso ◽  
Silvia Zorrilla ◽  
Marta Sobrinos-Sanguino ◽  
Miguel Ángel Robles-Ramos ◽  
Carlos Alfonso ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Lipid Membranes ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Content Type ◽  
E Coli ◽  
Daughter Cells ◽  
Z Ring

ABSTRACTDivision ring formation at midcell is controlled by various mechanisms inEscherichia coli, one of them being the linkage between the chromosomal Ter macrodomain and the Z-ring mediated by MatP, a DNA binding protein that organizes this macrodomain and contributes to the prevention of premature chromosome segregation. Here we show that, during cell division, just before splitting the daughter cells, MatP seems to localize close to the cytoplasmic membrane, suggesting that this protein might interact with lipids. To test this hypothesis, we investigated MatP interaction with lipidsin vitro. We found that, when encapsulated inside vesicles and microdroplets generated by microfluidics, MatP accumulates at phospholipid bilayers and monolayers matching the lipid composition in theE. coliinner membrane. MatP binding to lipids was independently confirmed using lipid-coated microbeads and biolayer interferometry assays, which suggested that the recognition is mainly hydrophobic. Interaction of MatP with the lipid membranes also occurs in the presence of the DNA sequences specifically targeted by the protein, but there is no evidence of ternary membrane/protein/DNA complexes. We propose that the association of MatP with lipids may modulate its spatiotemporal localization and its recognition of other ligands.IMPORTANCEThe division of anE. colicell into two daughter cells with equal genomic information and similar size requires duplication and segregation of the chromosome and subsequent scission of the envelope by a protein ring, the Z-ring. MatP is a DNA binding protein that contributes both to the positioning of the Z-ring at midcell and the temporal control of nucleoid segregation. Our integratedin vivoandin vitroanalysis provides evidence that MatP can interact with lipid membranes reproducing the phospholipid mixture in theE. coliinner membrane, without concomitant recruitment of the short DNA sequences specifically targeted by MatP. This observation strongly suggests that the membrane may play a role in the regulation of the function and localization of MatP, which could be relevant for the coordination of the two fundamental processes in which this protein participates, nucleoid segregation and cell division.

scholarly journals Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

2019 ◽  
Vol 20 (13) ◽  
pp. 3336 ◽  
Author(s):  
James Shell Cox ◽  
Kristi Moncja ◽  
Mykala Mckinnes ◽  
Michael W. Van Dyke
Keyword(s):  
Dna Binding ◽  
Massively Parallel Sequencing ◽  
Thermus Thermophilus ◽  
Recognition Sequence ◽  
Promoter Regions ◽  
Thermus Thermophilus Hb8 ◽  
Dna Binding Sites ◽  
Transcriptional Regulatory ◽  
Genes Encoding ◽  
Putative Transcription Factor

Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism’s transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5′–AACnAACGTTnGTT–3′ that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.

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