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.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

scholarly journals Interactions between the Promoter Regions of Nitrogenase Structural Genes (nifHDK2) and DNA-Binding Proteins from N2- and Ammonium-Grown Cells of the Archaeon Methanosarcina barkeri 227

1998 ◽  
Vol 180 (10) ◽  
pp. 2723-2728 ◽  
Author(s):  
Yueh-tyng Chien ◽  
John D. Helmann ◽  
Stephen H. Zinder
Keyword(s):  
Dna Binding ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Common Factor ◽  
Methanosarcina Barkeri ◽  
Promoter Regions ◽  
Putative Promoter Region ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Methyl Coenzyme M Reductase

ABSTRACT Transcription initiation in Archaea (archaebacteria) resembles the eucaryotic process, having been shown to involve TATA box-like promoter regions as well as TATA-binding protein and TFIIB homologs. However, little is known about transcription regulation in archaea. We have previously demonstrated that transcripts ofnifHDK2 genes, encoding Methanosarcina barkerinitrogenase, are present in N2-grown cells but not in ammonium-grown cells, indicating that nif transcription is regulated by the nitrogen source. In this study, we detected proteins in M. barkeri cell extracts that bind specifically to DNA containing the putative promoter region of nifHDK2. No binding was found when the promoter region was deleted from the DNA. A competition assay showed that the methyl coenzyme M reductase (mcr) promoter region DNA and the nifH2promoter region DNA competed for a common factor(s). There was no binding to the nifH2 promoter region by extracts of ammonium-grown cells, but there was binding by these extracts to promoter regions for mcr genes, which are presumably constitutively expressed. Interestingly, extracts of ammonium-grown cells inhibited binding to the nif promoter region by extracts of N2-grown cells. Fractionation of extracts of ammonium-grown cells with a heparin-Sepharose column resolved them into a fraction eluting at 0 M NaCl, which inhibited binding by extracts of N2-grown cells, and a fraction eluting at 0.5 to 0.75 M NaCl, which showed binding to the promoter region. These results are congruent with a model for regulation of nif gene expression in M. barkeri in which a substance present in ammonium-grown cells inhibits DNA binding by a transcription-associated protein or proteins.

scholarly journals Transcriptional Activities of the Zinc Finger Protein Zac Are Differentially Controlled by DNA Binding

2003 ◽  
Vol 23 (3) ◽  
pp. 988-1003 ◽  
Author(s):  
Anke Hoffmann ◽  
Elisabetta Ciani ◽  
Joel Boeckardt ◽  
Florian Holsboer ◽  
Laurent Journot ◽  
...  
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Zinc Fingers ◽  
Repeat Elements ◽  
Functional Connections ◽  
Finger Protein ◽  
Dna Binding Sites ◽  
Transcriptional Regulatory ◽  
Dna Elements

ABSTRACT Zac encodes a zinc finger protein that promotes apoptosis and cell cycle arrest and is maternally imprinted. Here, we show that Zac contains transactivation and repressor activities and that these transcriptional activities are differentially controlled by DNA binding. Zac transactivation mapped to two distinct domains. One of these contained multiple repeats of the peptide PLE, which behaved as an autonomous activation unit. More importantly, we identified two related high-affinity DNA-binding sites which were differentially bound by seven Zac C2H2 zinc fingers. Zac bound as a monomer through zinc fingers 6 and 7 to the palindromic DNA element to confer transactivation. In contrast, binding as a monomer to one half-site of the repeat element turned Zac into a repressor. Conversely, Zac dimerization at properly spaced direct and reverse repeat elements enabled transactivation, which strictly correlated with DNA-dependent and -independent contacts of key residues within the recognition helix of zinc finger 7. The later ones support specific functional connections between Zac DNA binding and transcriptional-regulatory surfaces. Both classes of DNA elements were identified in a new Zac target gene and confirmed that the zinc fingers communicate with the transactivation function. Together, our data demonstrate a role for Zac as a transcription factor in addition to its role as coactivator for nuclear receptors and p53.

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 Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

2004 ◽  
Vol 186 (22) ◽  
pp. 7659-7669 ◽  
Author(s):  
Astrid P. Koudelka ◽  
Lisa A. Hufnagel ◽  
Gerald B. Koudelka
Keyword(s):  
Dna Binding ◽  
Shiga Toxin ◽  
Binding Activity ◽  
Purification And Characterization ◽  
Gene Encoding ◽  
Dna Binding Activity ◽  
Transcriptional Regulatory ◽  
Genes Encoding ◽  
Lambdoid Prophage

ABSTRACT The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (OR). Also typical, when bound at OR, 933W repressor functions as an activator at the PRM promoter and a repressor at the PR promoter. In contrast to other lambdoid bacteriophages, 933W left operator (OL) contains only two repressor binding sites, but the OL-bound repressor still efficiently represses PL transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a “linker ” region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.

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.

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