scholarly journals Investigating the DNA-Binding Site for VirB, a Key Transcriptional Regulator of Shigella Virulence Genes, Using an In Vivo Binding Tool

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 149 ◽  
Author(s):  
Monika Karney ◽  
Joy McKenna ◽  
Natasha Weatherspoon-Griffin ◽  
Alexander Karabachev ◽  
Makensie Millar ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Inverted Repeat ◽  
Virulence Genes ◽  
In Vivo Studies ◽  
Single Nucleotide ◽  
Binding Interactions ◽  
Shigella Species ◽  
Dna Binding Site

The transcriptional anti-silencing and DNA-binding protein, VirB, is essential for the virulence of Shigella species and, yet, sequences required for VirB-DNA binding are poorly understood. While a 7-8 bp VirB-binding site has been proposed, it was derived from studies at a single VirB-dependent promoter, icsB. Our previous in vivo studies at a different VirB-dependent promoter, icsP, found that the proposed VirB-binding site was insufficient for regulation. Instead, the required site was found to be organized as a near-perfect inverted repeat separated by a single nucleotide spacer. Thus, the proposed 7-8 bp VirB-binding site needed to be re-evaluated. Here, we engineer and validate a molecular tool to capture protein-DNA binding interactions in vivo. Our data show that a sequence organized as a near-perfect inverted repeat is required for VirB-DNA binding interactions in vivo at both the icsB and icsP promoters. Furthermore, the previously proposed VirB-binding site and multiple sites found as a result of its description (i.e., sites located at the virB, virF, spa15, and virA promoters) are not sufficient for VirB to bind in vivo using this tool. The implications of these findings are discussed.

scholarly journals Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1.

1994 ◽  
Vol 14 (6) ◽  
pp. 3842-3852 ◽  
Author(s):  
C Cheng ◽  
N Kacherovsky ◽  
K M Dombek ◽  
S Camier ◽  
S K Thukral ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Inverted Repeat ◽  
Target Genes ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Potential Target

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.

scholarly journals VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

2020 ◽  
Author(s):  
Jillian N. Socea ◽  
Grant R. Bowman ◽  
Helen J. Wing
Keyword(s):  
Dna Binding ◽  
Subcellular Localization ◽  
Binding Site ◽  
Virulence Genes ◽  
Shigella Flexneri ◽  
Transcriptional Regulator ◽  
Virulence Plasmid ◽  
Dna Binding Site ◽  
Bacterial Cytoplasm ◽  
Insight Into

AbstractVirB is a key regulator of virulence genes located on the large virulence plasmid (pINV) of the bacterial pathogen Shigella flexneri. VirB is unusual in that it is not related to other transcriptional regulators, instead, it belongs to a protein family that primarily functions in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing of virulence genes mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in cells cured of pINV. Moreover, in the context of the fusion, amino acid substitutions in the DNA binding domain of VirB resulted in the fluorescent signal becoming entirely diffuse. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

scholarly journals One-dimensional diffusion of TrpR along DNA enhances its affinity for the operator by chemical ratchet mechanism

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takashi Kinebuchi ◽  
Nobuo Shimamoto
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Diffusion Mechanism ◽  
Detailed Balance ◽  
Dna Looping ◽  
Antenna Effect ◽  
One Dimensional ◽  
Dimensional Diffusion ◽  
Dna Binding Site

AbstractSeveral DNA-binding proteins show the affinities for their specific DNA sites that positively depend on the length of DNA harboring the sites, i. e. antenna effect. DNA looping can cause the effect for proteins with two or more DNA binding sites, i. e. the looping mechanism. One-dimensional diffusion also has been suggested to cause the effect for proteins with single DNA sites, the diffusion mechanism, which could violate detailed balance. We addressed which mechanism is possible for E. coli TrpR showing 104-fold antenna effect with a single DNA binding site. When a trpO-harboring DNA fragment was connected to a nonspecific DNA with biotin-avidin connection, the otherwise sevenfold antenna effect disappeared. This result denies the looping mechanism with an unknown second DNA binding site. The 3.5-fold repression by TrpR in vivo disappeared when a tight LexA binding site was introduced at various sites near the trpO, suggesting that the binding of LexA blocks one-dimensional diffusion causing the antenna effect. These results are consistent with the chemical ratchet recently proposed for TrpR-trpO binding to solve the deviation from detailed balance, and evidence that the antenna effect due to one-dimensional diffusion exists in cells.

scholarly journals Derivation and Functional Characterization of a Consensus DNA Binding Sequence for the Tas Transcriptional Activator of Simian Foamy Virus Type 1

1998 ◽  
Vol 72 (7) ◽  
pp. 5502-5509 ◽  
Author(s):  
Yibin Kang ◽  
Bryan R. Cullen
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Foamy Virus ◽  
Wild Type ◽  
Simian Foamy Virus ◽  
Internal Promoter ◽  
Dna Binding Site ◽  
Site Consensus

ABSTRACT Although DNA binding sites specific for the Bel-1 and Tas transcriptional activators, encoded, respectively, by the human and simian foamy viruses, have been mutationally defined, they show little evident sequence identity. As a result, the sequence determinants for DNA binding by both Bel-1 and Tas have remained unclear. Here, we report the use of a novel in vivo randomization and selection strategy to identify a Tas DNA binding site consensus. This approach takes advantage of the fact that Tas can effectively activate gene expression in yeast cells via a Tas DNA binding site derived from the simian foamy virus type 1 (SFV-1) internal promoter. The defined Tas DNA binding site consensus extends over approximately 25 bp and contains a critical core sequence of ∼5 bp. Positions adjacent to this core sequence, while clearly also subject to selection, show a significantly higher level of sequence variation. Surprisingly, the wild-type SFV-1 internal promoter Tas DNA binding site fails to conform to the consensus at several positions. Further analysis demonstrated that the consensus sequence bound Tas more effectively than did the wild-type sequence in vitro and could mediate an enhanced Tas response in vivo when substituted into the SFV-1 internal promoter context. These findings explain the limited sequence identity observed for mutationally defined Tas or Bel-1 response elements and should facilitate the identification of Tas DNA target sites located elsewhere in the SFV-1 genome.

scholarly journals Identification and Functional Characterization of a High-Affinity Bel-1 DNA Binding Site Located in the Human Foamy Virus Internal Promoter

1998 ◽  
Vol 72 (1) ◽  
pp. 504-511 ◽  
Author(s):  
Yibin Kang ◽  
Wade S. Blair ◽  
Bryan R. Cullen
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Foamy Virus ◽  
Promoter Element ◽  
Internal Promoter ◽  
Dna Binding Site ◽  
Foamy Viruses ◽  
Ltr Promoter

ABSTRACT The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3′ end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902–3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.

Identification of potential target genes for Adr1p through characterization of essential nucleotides in UAS1

1994 ◽  
Vol 14 (6) ◽  
pp. 3842-3852
Author(s):  
C Cheng ◽  
N Kacherovsky ◽  
K M Dombek ◽  
S Camier ◽  
S K Thukral ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Inverted Repeat ◽  
Target Genes ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Potential Target

Adr1p is a regulatory protein in the yeast Saccharomyces cerevisiae that binds to and activates transcription from two sites in a perfect 22-bp inverted repeat, UAS1, in the ADH2 promoter. Binding requires two C2H2 zinc fingers and a region amino terminal to the fingers. The importance for DNA binding of each position within UAS1 was deduced from two types of assays. Both methods led to an identical consensus sequence containing only four essential base pairs: GG(A/G)G. The preferred sequence, TTGG(A/G)GA, is found in both halves of the inverted repeat. The region of Adr1p amino terminal to the fingers is important for phosphate contacts in the central region of UAS1. However, no base-specific contacts in this portion of UAS1 are important for DNA binding or for ADR1-dependent transcription in vivo. When the central 6 bp were deleted, only a single monomer of Adr1p was able to bind in vitro and activation in vivo was severely reduced. On the basis of these results and previous knowledge about the DNA binding site requirements, including constraints on the spacing and orientation of sites that affect activation in vivo, a consensus binding site for Adr1p was derived. By using this consensus site, potential Adr1p binding sites were located in the promoters of genes known to show ADR1-dependent expression. In addition, this consensus allowed the identification of new potential target genes for Adr1p.

Sequence-specific transcriptional activation by Myc and repression by Max

1993 ◽  
Vol 13 (1) ◽  
pp. 383-390
Author(s):  
C Amin ◽  
A J Wagner ◽  
N Hay
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Leucine Zipper ◽  
Cellular Proliferation ◽  
Helix Loop Helix ◽  
Dna Binding Site ◽  
Carboxy Terminal

The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription factors. However, the mechanism of its action in vivo is not yet clear. The discovery of the specific cognate DNA-binding site for Myc and its specific heterodimerization partner, Max, enabled the use of direct experiments to elucidate how Myc functions in vivo and how this function is modulated by Max. Here we demonstrate that exogenously expressed Myc is capable of activating transcription in vivo through its specific DNA-binding site. Moreover, transcriptional activation by Myc is dependent on the basic region, the integrity of the helix-loop-helix and leucine zipper dimerization motifs located in the carboxy-terminal portion of the protein, and the regions in the amino terminus conserved among Myc family proteins. In contrast to Myc, exogenously expressed Max elicited transcriptional repression and blocked transcriptional activation by Myc through the same DNA-binding site. Our results suggest a functional antagonism between Myc and Max which is mediated by their relative levels in the cells. A model for the activity of Myc and Max in vivo is presented.

scholarly journals Sequence-specific transcriptional activation by Myc and repression by Max.

1993 ◽  
Vol 13 (1) ◽  
pp. 383-390 ◽  
Author(s):  
C Amin ◽  
A J Wagner ◽  
N Hay
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Leucine Zipper ◽  
Cellular Proliferation ◽  
Helix Loop Helix ◽  
Dna Binding Site ◽  
Carboxy Terminal

The c-Myc oncoprotein, which is required for cellular proliferation, resembles in its structure a growing number of transcription factors. However, the mechanism of its action in vivo is not yet clear. The discovery of the specific cognate DNA-binding site for Myc and its specific heterodimerization partner, Max, enabled the use of direct experiments to elucidate how Myc functions in vivo and how this function is modulated by Max. Here we demonstrate that exogenously expressed Myc is capable of activating transcription in vivo through its specific DNA-binding site. Moreover, transcriptional activation by Myc is dependent on the basic region, the integrity of the helix-loop-helix and leucine zipper dimerization motifs located in the carboxy-terminal portion of the protein, and the regions in the amino terminus conserved among Myc family proteins. In contrast to Myc, exogenously expressed Max elicited transcriptional repression and blocked transcriptional activation by Myc through the same DNA-binding site. Our results suggest a functional antagonism between Myc and Max which is mediated by their relative levels in the cells. A model for the activity of Myc and Max in vivo is presented.

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