scholarly journals Distinct Roles of Two Binding Sites for the Bovine Papillomavirus (BPV) E2 Transactivator on BPV DNA Replication

1998 ◽  
Vol 72 (7) ◽  
pp. 5735-5744 ◽  
Author(s):  
Thomas G. Gillette ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
High Mobility ◽  
Bovine Papillomavirus ◽  
Mobility Shift ◽  
Distal Edge ◽  
Lower Mobility ◽  
Dyad Symmetry ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.

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.

NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene

2008 ◽  
Vol 86 (1) ◽  
pp. 46-56 ◽  
Author(s):  
José R. Blesa ◽  
Jesús A. Prieto-Ruiz ◽  
Beth A. Abraham ◽  
Bridget L. Harrison ◽  
Anita A. Hegde ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Colorectal Tumors ◽  
Methylation Analysis ◽  
Mobility Shift ◽  
Nuclear Respiratory Factors ◽  
Hela S3 ◽  
Electrophoretic Mobility Shift Assays

The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear–mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5′ region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity.

scholarly journals Orientation of a Novel DNA Binding Site Affects Human Papillomavirus-Mediated Transcription and Replication

2001 ◽  
Vol 75 (4) ◽  
pp. 1722-1735 ◽  
Author(s):  
Christopher D. Newhouse ◽  
Saul J. Silverstein
Keyword(s):  
Human Papillomavirus ◽  
Dna Replication ◽  
Binding Site ◽  
Dna Sequences ◽  
Binding Sites ◽  
Long Control Region ◽  
Bovine Papillomavirus ◽  
The Novel ◽  
Consensus Binding Site ◽  
Mobility Shift

ABSTRACT A consensus binding site for the human papillomavirus (HPV) E2 protein was determined from an unbiased set of degenerate oligonucleotides using cyclic amplification and selection of targets (CASTing). Detectable DNA-protein complexes were formed after six to nine cycles of CASTing. A population of selected binding sites was cloned, and a consensus was determined by statistical analysis of the DNA sequences of individual isolates. Starting from a pool with 20 random bases, a consensus binding site of ACAC-N5-GGT was derived. CASTing and electrophoretic mobility shift analyses demonstrate that human but not bovine papillomavirus E2 proteins recognize this sequence. The presence of this sequence in papillomavirus genomes suggests a role for its function. We demonstrate that this site functionally substitutes for the canonical E2 binding site (ACCG-N4-CGGT) in both transient-transcription and DNA replication assays. This sequence, in most instances, is interchangeable with the resident E2 binding sites in the context of the HPV type 16 long control region. Where the novel sequence does not support E2-mediated effects on gene expression or DNA replication, we demonstrate that changing the orientation of the novel sequence restores this effect.

scholarly journals CtrA, a Global Response Regulator, Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus

2009 ◽  
Vol 191 (17) ◽  
pp. 5458-5470 ◽  
Author(s):  
William Spencer ◽  
Rania Siam ◽  
Marie-Claude Ouimet ◽  
D. Patrick Bastedo ◽  
Gregory T. Marczynski
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Cell Cycle Control ◽  
Transcription Control ◽  
Mobility Shift ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [Kd ], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (Kd , >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.

scholarly journals Control of Directionality in Bacteriophage mv4 Site-Specific Recombination: Functional Analysis of the Xis Factor

2009 ◽  
Vol 192 (3) ◽  
pp. 624-635 ◽  
Author(s):  
Michèle Coddeville ◽  
Paul Ritzenthaler
Keyword(s):  
Binding Sites ◽  
Basic Protein ◽  
Lactobacillus Delbrueckii ◽  
Mobility Shift ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dna Binding Sites ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the host attB site during Lactobacillus delbrueckii lysogenization. The mv4 prophage is excised during the induction of lytic growth. Excisive site-specific recombination between the attR and attL sites is also catalyzed by the phage-encoded recombinase, but the directionality of the recombination is determined by a second phage-encoded protein, the recombination directionality factor (RDF). We have identified and functionally characterized the RDF involved in site-specific excision of the prophage genome. The mv4 RDF, mv4Xis, is encoded by the second gene of the early lytic operon. It is a basic protein of 56 amino acids. Electrophoretic mobility shift assays demonstrated that mv4Xis binds specifically to the attP and attR sites via two DNA-binding sites, introducing a bend into the DNA. In vitro experiments and in vivo recombination assays with plasmids in Escherichia coli and Lactobacillus plantarum demonstrated that mv4Xis is absolutely required for inter- or intramolecular recombination between the attR and attL sites. In contrast to the well-known phage site-specific recombination systems, the integrative recombination between the attP and attB sites seems not to be inhibited by the presence of mv4Xis.

scholarly journals Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2749-2756 ◽  
Author(s):  
Nisheeth Agarwal ◽  
Tirumalai R. Raghunand ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Site ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Mobility Shift ◽  
Camp Analogue ◽  
Camp Receptor ◽  
Fusion Analysis ◽  
Electrophoretic Mobility Shift Assays

The wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at −58.5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRPM (encoded by the M. tuberculosis gene Rv3676) to the whiB1 5′ untranslated region (5′UTR). β-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRPM to a consensus CRP-binding site in the whiB1 5′UTR.

scholarly journals ApoFnr Binds as a Monomer to Promoters Regulating the Expression of Enterotoxin Genes of Bacillus cereus

2008 ◽  
Vol 190 (12) ◽  
pp. 4242-4251 ◽  
Author(s):  
Julia Esbelin ◽  
Yves Jouanneau ◽  
Jean Armengaud ◽  
Catherine Duport
Keyword(s):  
Fusion Protein ◽  
Bacillus Cereus ◽  
Regulatory Protein ◽  
Disulfide Bridge ◽  
Oligomeric State ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Enterotoxin Genes ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Bacillus cereus Fnr is a member of the Crp/Fnr (cyclic AMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of hbl and nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occurred as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulfide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE, and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed.

scholarly journals Replication from oriP of Epstein-Barr Virus Requires Exact Spacing of Two Bound Dimers of EBNA1 Which Bend DNA

2001 ◽  
Vol 75 (22) ◽  
pp. 10603-10611 ◽  
Author(s):  
Jacqueline M. Bashaw ◽  
John L. Yates
Keyword(s):  
Electrophoretic Mobility ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Mobility Shift ◽  
Barr Virus ◽  
Cellular Factors ◽  
Specific Nucleotide Sequence ◽  
Epstein Barr ◽  
Stable Maintenance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriPis an ∼120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.

scholarly journals Binding of Gal4p and Bicoid to Nucleosomal Sites in Yeast in the Absence of Replication

1999 ◽  
Vol 19 (4) ◽  
pp. 2977-2985 ◽  
Author(s):  
Bhuvana Balasubramanian ◽  
Randall H. Morse
Keyword(s):  
Transcription Factors ◽  
Dna Replication ◽  
Binding Sites ◽  
Transcriptional Activator ◽  
Nucleosome Positioning ◽  
Living Cells ◽  
Intracellular Environment ◽  
Nucleosomal Dna

ABSTRACT The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with α-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of theDrosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to nucleosomal sites inSWI + and swi1Δ yeast and in the absence of replication. Our results indicate that some feature of the intracellular environment other than DNA replication or the SWI-SNF complex permits factor access to nucleosomal sites.

scholarly journals Functional Characterization of a Novel Ku70/80 Pause Site at the H19/Igf2 Imprinting Control Region

2005 ◽  
Vol 25 (10) ◽  
pp. 3855-3863 ◽  
Author(s):  
David J. Katz ◽  
Michael A. Beer ◽  
John M. Levorse ◽  
Shirley M. Tilghman
Keyword(s):  
Binding Sites ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Binding Activity ◽  
Mobility Shift ◽  
Control Center ◽  
Dna Binding Activity ◽  
A Genome ◽  
Pause Site

ABSTRACT The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg2+, implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

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