Amplified probing of protein/DNA interactions for sensitive fluorescence detection of transcription factors

2018 ◽  
Vol 6 (37) ◽  
pp. 6002-6007 ◽  
Author(s):  
Jin Li ◽  
Jinshan Tang ◽  
Bingying Jiang ◽  
Yun Xiang ◽  
Ruo Yuan
Keyword(s):  
Transcription Factors ◽  
Fluorescence Detection ◽  
Metal Ion ◽  
Dna Interactions ◽  
Protection Strategy ◽  
Protein Dna Interactions ◽  
Enzyme Protection

Coupling the enzyme protection strategy with metal-ion dependent DNAzyme amplification leads to sensitive monitoring of protein/DNA interactions.

scholarly journals HcaR Ligand and DNA Interactions in the Regulation of Catabolic Gene Expression

2014 ◽  
Vol 70 (a1) ◽  
pp. C203-C203
Author(s):  
Andrzej Joachimiak ◽  
Grazyna Joachimiak ◽  
Lance Bigelow ◽  
Garrett Cobb ◽  
Youngchang Kim
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Binding ◽  
Aromatic Compounds ◽  
Environmental Research ◽  
Structural Basis ◽  
Dna Interactions ◽  
Catabolic Genes ◽  
Protein Dna Interactions ◽  
Acinetobacter Sp

Precise tuning of gene expression by transcriptional regulators determines the response to internal and external chemical signals and adjusts the metabolic machinery for many cellular processes. As a part of ongoing efforts by the Midwest Center for Structural Genomics, a number of transcription factors were selected to study protein-ligand and protein-DNA interactions. HcaR, a new member of the MarR/SlyA family of transcription regulators from soil bacteria Acinetobacter sp. ADP1, is an evolutionarily atypical regulator and represses hydroxycinnamate (hca) catabolic genes. Hydroxycinnamates containing an aromatic ring play diverse, critical roles in plant architecture and defense. HcaR regulates the expression of the hca catabolic operon, allowing this and related bacterial strains to utilize hydroxycinnamates: ferulate, p-coumarate, and caffeate as sole sources of carbon and energy. HcaR appears to be capable of responding to multiple aromatic ligands. These aromatic compounds bind to HcaR and reduce its affinity to the specific DNA sites. As a result, the transcription of genes encoding several catabolic enzymes is up-regulated. The HcaR structures of the apo-form and in a complex with several ligands: ferulic acid, 3,4 dihydroxybenzoic acid, vanillin and p-coumaric acid have been determined to understand how HcaR accommodates various aromatic compounds using the same binding pocket. We also have identified a potential DNA site for HcaR in the regulatory region upstream of the genes of the hca catabolic operon in Acinetobacter sp. ADP1 and have confirmed DNA binding by EMSA. The co-crystal structure of HcaR and palindromic 24-mer DNA has been determined for this DNA site. The crystal structures of HcaR, the apo-form, ligand-bound forms, and the specific DNA-bound form provide critical structural basis of protein-ligand (substrates or product) and protein-DNA interactions to understand the regulation of the expression of hydroxycinnamate (hca) catabolic genes. Our studies allow for better understanding of DNA-binding and regulation by this important group of transcription factors belonging to the MarR/SlyA families. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Small-molecule inhibitors of dimeric transcription factors: Antagonism of protein–protein and protein–DNA interactions

MedChemComm ◽  
2012 ◽  
Vol 3 (5) ◽  
pp. 541 ◽  
Author(s):  
Jeremy L. Yap ◽  
Jay Chauhan ◽  
Kwan-Young Jung ◽  
Lijia Chen ◽  
Edward V. Prochownik ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Dna Interactions ◽  
Protein Dna Interactions

scholarly journals Universal correction of enzymatic sequence bias reveals molecular signatures of protein/DNA interactions

2017 ◽  
Author(s):  
André L. Martins ◽  
Ninad M. Walavalkar ◽  
Warren D. Anderson ◽  
Chongzhi Zang ◽  
Michael J. Guertin
Keyword(s):  
Experimental Data ◽  
Transcription Factors ◽  
Nucleic Acid ◽  
High Throughput Sequencing ◽  
Molecular Signatures ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Resolution Mapping ◽  
Nucleic Acid Interactions ◽  
Sequence Bias

AbstractCoupling molecular biology to high throughput sequencing has revolutionized the study of biology. Molecular genomics techniques are continually refined to provide higher resolution mapping of nucleic acid interactions and structure. Sequence preferences of enzymes can interfere with the accurate interpretation of these data. We developed seqOutBias to characterize enzymatic sequence bias from experimental data and scale individual sequence reads to correct intrinsic enzymatic sequence biases. SeqOutBias efficiently corrects DNase-seq, TACh-seq, ATAC-seq, MNase-seq, and PRO-seq data. We show that seqOutBias correction facilitates identification of true molecular signatures resulting from transcription factors and RNA polymerase interacting with DNA.

scholarly journals Crystal structure of the Wheat dwarf virus Rep domain

2019 ◽  
Vol 75 (12) ◽  
pp. 744-749 ◽  
Author(s):  
Blake A. Everett ◽  
Lauren A. Litzau ◽  
Kassidy Tompkins ◽  
Ke Shi ◽  
Andrew Nelson ◽  
...  
Keyword(s):  
Metal Ion ◽  
Initial Step ◽  
Dwarf Virus ◽  
Sequence Specificity ◽  
Wheat Dwarf Virus ◽  
Rolling Circle Replication ◽  
Dna Interactions ◽  
Subsequent Formation ◽  
Rolling Circle ◽  
Protein Dna Interactions

The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein–ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein–DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24 Å resolution and represents an initial step in the direction of solving the structure of a protein–ssDNA complex.

scholarly journals An improved bind-n-seq strategy to determine protein-DNA interactions validated using the bacterial transcriptional regulator YipR

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Shi-qi An ◽  
Miguel A. Valvano ◽  
Yan-hua Yu ◽  
Jeremy S. Webb ◽  
Guillermo Lopez Campos
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Computational Algorithm ◽  
Dna Recombination ◽  
Transcriptional Regulator ◽  
Sequence Motif ◽  
Dependent Manner ◽  
Dna Interactions ◽  
Promoter Regions ◽  
Protein Dna Interactions

Abstract Background Interactions between transcription factors and DNA lie at the centre of many biological processes including DNA recombination, replication, repair and transcription. Most bacteria encode diverse proteins that act as transcription factors to regulate various traits. Several technologies for identifying protein–DNA interactions at the genomic level have been developed. Bind-n-seq is a high-throughput in vitro method first deployed to analyse DNA interactions associated with eukaryotic zinc-finger proteins. The method has three steps (i) binding protein to a randomised oligonucleotide DNA target library, (ii) deep sequencing of bound oligonucleotides, and (iii) a computational algorithm to define motifs among the sequences. The classical Bind-n-seq strategy suffers from several limitations including a lengthy wet laboratory protocol and a computational algorithm that is difficult to use. We introduce here an improved, rapid, and simplified Bind-n-seq protocol coupled with a user-friendly downstream data analysis and handling algorithm, which has been optimized for bacterial target proteins. We validate this new protocol by showing the successful characterisation of the DNA-binding specificities of YipR (YajQ interacting protein regulator), a well-known transcriptional regulator of virulence genes in the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc). Results The improved Bind-n-seq approach identified several DNA binding motif sequences for YipR, in particular the CCCTCTC motif, which were located in the promoter regions of 1320 Xcc genes. Informatics analysis revealed that many of these genes regulate functions associated with virulence, motility, and biofilm formation and included genes previously found involved in virulence. Additionally, electromobility shift assays show that YipR binds to the promoter region of XC_2633 in a CCCTCTC motif-dependent manner. Conclusion We present a new and rapid Bind-n-seq protocol that should be useful to investigate DNA-binding proteins in bacteria. The analysis of YipR DNA binding using this protocol identifies a novel DNA sequence motif in the promoter regions of target genes that define the YipR regulon.

scholarly journals A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2

2021 ◽  
Vol 118 (11) ◽  
pp. e2012835118
Author(s):  
Kyle D. Pedro ◽  
Luis M. Agosto ◽  
Jared A. Sewell ◽  
Kimberly A. Eberenz ◽  
Xianbao He ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Computational Modeling ◽  
High Throughput ◽  
Molecular Networks ◽  
Transcriptional Networks ◽  
Dna Interactions ◽  
Long Terminal Repeats ◽  
Functional Screen ◽  
Protein Dna Interactions ◽  
Hiv 1

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein–DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein–DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.

scholarly journals Prediction of protein-DNA interactions of transcription factors linking proteomics and transcriptomics data

2016 ◽  
Vol 13 ◽  
pp. 14-23 ◽  
Author(s):  
Yu. Kondrakhin ◽  
T. Valeev ◽  
R. Sharipov ◽  
I. Yevshin ◽  
F. Kolpakov ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Transcriptomics Data

scholarly journals An improved ChEC-seq method accurately maps the genome-wide binding of transcription coactivators and sequence-specific transcription factors

2021 ◽  
Author(s):  
Rafal Donczew ◽  
Amélia Lalou ◽  
Didier Devys ◽  
Laszlo Tora ◽  
Steven Hahn
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Sequence ◽  
Dna Interactions ◽  
Experimental Conditions ◽  
Genome Wide ◽  
Protein Dna Interactions ◽  
Cleavage Step ◽  
Computational Analyses ◽  
Active Genes

AbstractMittal and colleagues have raised questions about mapping transcription factor locations on DNA using the MNase-based ChEC-seq method (Mittal et al., 2021). Partly due to this concern, we modified the experimental conditions of the MNase cleavage step and subsequent computational analyses, resulting in more stringent conditions for mapping protein-DNA interactions (Donczew et al., 2020). The revised method (dx.doi.org/10.17504/protocols.io.bizgkf3w) answers questions raised by Mittal et al. and, without changing earlier conclusions, identified widespread promoter binding of the transcription coactivators TFIID and SAGA at active genes. The revised method is also suitable for accurately mapping the genome-wide locations of DNA sequence-specific transcription factors.

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