scholarly journals Mapping protein binding stability on nucleosomal DNA by single-molecule approach

2014 ◽  
Vol 70 (a1) ◽  
pp. C111-C111
Author(s):  
Jianshi Jin ◽  
Teng-fei Lian ◽  
Xiaoliang Xie ◽  
Xiao-Dong Su
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Binding Sites ◽  
Binding Domain ◽  
Shielding Effect ◽  
Dna Binding Domain ◽  
Nucleosomal Dna ◽  
Dna Protein Interaction

The conformation of nucleosomal DNA is significantly different from that of a canonical B-form double stranded DNA (dsDNA), and is generally regarded to be less flexible and less accessible than free dsDNA due to the tight association of histone cores. Previous studies have demonstrated that the key mechanism involved in nucleosomal DNA-protein interaction is the protein accessibility to the DNA binding site. In this work, we used single molecule assays to measure the stability of two transcriptional factors (glucocorticoid receptor DNA binding domain (GRDBD) and estrogen receptor DNA-binding domain (ERDBD)) bound to their binding sites on different positions of the nucleosomal DNA. Interestingly, the results demonstrated that the nucleosomal DNA-GRDBD binding is not always consistent with the histone shielding effect, but adjusted by additional structural changes. Furthermore, the changes of these DNA-GRDBD interaction profiles were confirmed using molecular modeling and docking approaches based on their crystal structures. Very differently, ERDBD essentially is unable to bind to the nucleosomal DNA anywhere including the unblocked positions. We thus have concluded that the nucleosomal DNA-protein interaction is regulated not only by the histone shielding of the DNA binding sites, but also by the conformational changes of the nucleosomal DNA.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

2007 ◽  
Vol 403 (3) ◽  
pp. 463-472 ◽  
Author(s):  
Nathalie Gillard ◽  
Stephane Goffinont ◽  
Corinne Buré ◽  
Marie Davidkova ◽  
Jean-Claude Maurizot ◽  
...  
Keyword(s):  
Dna Binding ◽  
Conformational Changes ◽  
Specific Binding ◽  
Classical Model ◽  
Binding Domain ◽  
Oh Radicals ◽  
Water Radiolysis ◽  
Dna Binding Domain ◽  
Fluorescence Measurements ◽  
Radiation Induced

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.

scholarly journals Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing.

1992 ◽  
Vol 12 (3) ◽  
pp. 1209-1217 ◽  
Author(s):  
C F Hardy ◽  
D Balderes ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Binding Protein ◽  
Binding Domain ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Carboxy Terminal

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Probing protein - DNA interaction by single molecule and structural analysis

2014 ◽  
Vol 70 (a1) ◽  
pp. C198-C198
Author(s):  
Jianshi Jin ◽  
Tengfei Lian ◽  
Chan Gu ◽  
Yiqin Gao ◽  
Yujie Sun ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Fundamental Property ◽  
Dna Interaction ◽  
Allosteric Effect ◽  
Dna Protein Interaction ◽  
Fine Tune

In proteins, conformational change impacting their function has been well investigated in the past decades, and was named `allosteric effect'. However, in DNA-protein interaction, the concept of DNA conformational change caused by DNA-protein binding will affect another nearby DNA-binding protein has not been well investigated and understood. Combined with structural biology and Single Molecule Assays, we can now probe and study allosteric propagation through DNA which exists as a fundamental property in DNA-protein interaction, and this allosteric effect through DNA can fine tune gene expression. Therefore, DNA conformational changes should be seriously considered and analyzed for DNA –protein interactions in general.

scholarly journals The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells

2007 ◽  
Vol 81 (12) ◽  
pp. 6254-6264 ◽  
Author(s):  
Stacy M. Horner ◽  
Daniel DiMaio
Keyword(s):  
Human Papillomavirus ◽  
Dna Binding ◽  
Binding Site ◽  
Cancer Cells ◽  
Binding Sites ◽  
Binding Domain ◽  
Carcinoma Cells ◽  
Dna Binding Domain ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells.

scholarly journals Single-molecule dynamics and genome-wide transcriptomics reveal that NF-κB (p65)-DNA binding times can be decoupled from transcriptional activation

2018 ◽  
Author(s):  
Andrea Callegari ◽  
Christian Sieben ◽  
Alexander Benke ◽  
David M. Suter ◽  
Beat Fierz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Single Molecule ◽  
Protein Interaction ◽  
Transcriptional Activation ◽  
Dna Binding Domain ◽  
Protein Protein Interaction ◽  
Genome Wide ◽  
Binding Time ◽  
Transcriptional Output

AbstractTranscription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.Author SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-κB (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

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