Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 813-823 ◽  
Author(s):  
Elizabeth L. Andersen Rawlinson ◽  
Ingolf F. Nes ◽  
Morten Skaugen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Direct Repeats ◽  
Mobility Shift ◽  
Dna Binding Site ◽  
Intergenic Dna ◽  
Dna Fragment ◽  
Derivatives Of

LasX regulates the transcription of the divergent operons lasXY and lasA–W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA–lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P lasA–W , and a third imperfect copy of this motif, overlapping the −10 element of P lasA–W , were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.

Molecular characterization of the CmbR activator-binding site in the metC–cysK promoter region in Lactococcus lactis

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Natasa Golic ◽  
Martijn Schliekelmann ◽  
María Fernández ◽  
Michiel Kleerebezem ◽  
Richard van Kranenburg
Keyword(s):  
Binding Site ◽  
Lactococcus Lactis ◽  
Promoter Region ◽  
Direct Repeat ◽  
Inverted Repeats ◽  
Sulphur Metabolism ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Gel Mobility Shift

The metC–cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the metC promoter. This binding was stimulated by O-acetyl-l-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

2000 ◽  
Vol 74 (5) ◽  
pp. 2084-2093 ◽  
Author(s):  
Joel Schaley ◽  
Robert J. O'Connor ◽  
Laura J. Taylor ◽  
Dafna Bar-Sagi ◽  
Patrick Hearing
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transient Expression ◽  
Binding Sites ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Protein Accumulation ◽  
Promoter Regions ◽  
Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

scholarly journals Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659 ◽  
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

scholarly journals Trans-activation of the human SOX3 promoter by MAZ in NT2/D1 cells

2008 ◽  
Vol 60 (3) ◽  
pp. 379-387 ◽  
Author(s):  
Natasa Kovacevic-Grujicic ◽  
Kazunari Yokoyama ◽  
Milena Stevanovic
Keyword(s):  
Gene Expression ◽  
Neuronal Differentiation ◽  
Gene Transcription ◽  
Binding Sites ◽  
Promoter Activity ◽  
Zinc Finger Protein ◽  
Mobility Shift ◽  
Finger Protein ◽  
Sox3 Gene

In this study, we examine the role of three highly conserved putative binding sites for Myc-associated zinc finger protein (MAZ) in regulation of the human SOX3 gene expression. Electrophoretic mobility shift and supershift assays indicate that complexes formed at two out of three MAZ sites of the human SOX3 promoter involve ubiquitously expressed MAZ protein. Furthermore, in cotransfection experiments we demonstrate that MAZ acts as a positive regulator of SOX3 gene transcription in both undifferentiated and RA-differentiated NT2/D1 cells. Although MAZ increased both basal and RA-induced promoter activity, our results suggest that MAZ does not contribute to RA inducibility of the SOX3 promoter during neuronal differentiation of NT2/D1 cells.

Distinct functional contributions of 2 GABP–NRF-2 recognition sites within the context of the human TOMM70 promoter

2006 ◽  
Vol 84 (5) ◽  
pp. 813-822 ◽  
Author(s):  
José R. Blesa ◽  
José Hernández-Yago
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Outer Mitochondrial Membrane ◽  
Mobility Shift ◽  
A Subunit ◽  
Expression Evidence ◽  
Electrophoretic Mobility Shift Assays

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported 2 binding sites for the transcription factor GABP–NRF-2 in the promoter region of the human TOMM70 gene that are important in activating transcription. To assess the functionality and actual role of these sites, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays were carried out. We conclude that GABP–NRF-2 binds in vivo to the TOMM70 promoter, and that the 2 GABP–NRF-2 binding sites of the promoter have different functional contributions in promoting TOMM70 expression. Evidence is provided that they work in an additive manner as single sites.

scholarly journals RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

2015 ◽  
Vol 197 (23) ◽  
pp. 3666-3675 ◽  
Author(s):  
Mei G. Lei ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Mobility Shift ◽  
Content Type ◽  
Virulence Regulation ◽  
Important Virulence Factor ◽  
Dna Fragment ◽  
Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

scholarly journals DNA Binding Site Selection of Dimeric and Tetrameric Stat5 Proteins Reveals a Large Repertoire of Divergent Tetrameric Stat5a Binding Sites

2000 ◽  
Vol 20 (1) ◽  
pp. 389-401 ◽  
Author(s):  
Elisabetta Soldaini ◽  
Susan John ◽  
Stefano Moro ◽  
Julie Bollenbacher ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Site Selection ◽  
Binding Sites ◽  
Tetramer Binding ◽  
Dna Binding Site ◽  
Potential Binding ◽  
Wide Range ◽  
Large Repertoire ◽  
Selection Of

ABSTRACT We have defined the optimal binding sites for Stat5a and Stat5b homodimers and found that they share similar core TTC(T/C)N(G/A)GAA interferon gamma-activated sequence (GAS) motifs. Stat5a tetramers can bind to tandemly linked GAS motifs, but the binding site selection revealed that tetrameric binding also can be seen with a wide range of nonconsensus motifs, which in many cases did not allow Stat5a binding as a dimer. This indicates a greater degree of flexibility in the DNA sequences that allow binding of Stat5a tetramers than dimers. Indeed, in an oligonucleotide that could bind both dimers and tetramers, it was possible to design mutants that affected dimer binding without affecting tetramer binding. A spacing of 6 bp between the GAS sites was most frequently selected, demonstrating that this distance is favorable for Stat5a tetramer binding. These data provide insights into tetramer formation by Stat5a and indicate that the repertoire of potential binding sites for this transcription factor is broader than expected.

The role of NeuroD1 in the upregulation of SMYD3 by HBx.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 183-183
Author(s):  
Junyao Xu ◽  
Qingqi Hong ◽  
Chuanchao He ◽  
Jie Wang
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Promoter Region ◽  
Gene Transfection ◽  
Histone Methyltransferase ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Cis Element ◽  
E Box

183 Background: SET and MYND Domain-Containing Protein 3 (SMYD3) is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis B virus x protein (HBx) is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Methods: Immunohistochemical staining was used to detect the expression of HBx and SMYD3 in HCC tumor tissues. HBx gene transfection, RNAi, and histone methyltransferase(H3-K4) activity assay were performed to reveal the transcrpitionally activation of HBx on functional SMYD3 gene expression. Chromatin immunoprecipitation (ChIP), Co-immunoprecipitation (Co-IP), Electrophoretic mobility shift assay (EMSA) were applied to investigate the underlying mechanism. Dual-luciferase reporter assay was used to search for the HBx responsive cis-element of SMYD3 gene. Results: Immunohistochemistry identified the positive correlation between HBx and SMYD3 expression in 42 HCC tissues. Up-regulation of HBx on SMYD3 expression was validated through experiments involving overexpression or knock-down of HBx in different HCC cell lines. And up-regulated SMYD3 is functionally active as histone methyltransferase. Next we found that HBx transcriptionally regulated SMYD3 gene expression by interacting with RNA polymerase IIand altering its binding site to a proximal promoter region(SD2) from a distant promoter region(SD6) of SMYD3. Truncated and mutant reporter assays revealed that the cis-element mapped in -178~-203bp in SMYD3 promotor is responsive for HBx-transactivation. And this 25bp cis-element contains a E-box 3 unit, which is a binding site for the transcriptional factor Neurogenic differentiation 1(NeuroD1). EMSA and Chip showed that HBx increased NeuroD1 binding to SMYD3 proximal promotor, however transcient expression of antisense NeuroD1 abolished HBx-induced SMYD3 expression. Conclusions: HBx transcriptionally up-regulates SMYD3 and that this process is mediated by NeuroD1 through binding to the E-box 3 site of SMYD3 promotor.

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