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.

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 An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

2009 ◽  
Vol 191 (17) ◽  
pp. 5489-5498 ◽  
Author(s):  
Steve D. Seredick ◽  
Barbara M. Seredick ◽  
David Baker ◽  
George B. Spiegelman
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Molecular Basis ◽  
Response Regulator ◽  
Mutant Protein ◽  
Wild Type ◽  
Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

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.

Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter

1994 ◽  
Vol 14 (10) ◽  
pp. 6607-6615
Author(s):  
E Neuman ◽  
E K Flemington ◽  
W R Sellers ◽  
W G Kaelin
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Mrna Abundance ◽  
Dna Binding Sites ◽  
Cell Cycle Dependence ◽  
Transcription Factor E2f ◽  
Cycle Dependent

The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance. The E2F-1 promoter contains four potential E2F-binding sites organized as two imperfect palindromes. Gel shift and transactivation studies suggested that these sites can bind to E2F in vitro and in vivo. Mutation of the two E2F palindromes abolished the cell cycle dependence of the E2F-1 promoter. Thus, E2F-1 appears to be regulated at the level of transcription, and this regulation is due, at least in part, to binding of one or more E2F family members to the E2F-1 promoter.

scholarly journals Temporal Regulation of Genes Encoding the Flagellar Proximal Rod in Caulobacter crescentus

2001 ◽  
Vol 183 (2) ◽  
pp. 725-735 ◽  
Author(s):  
Charles H. Boyd ◽  
James W. Gober
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Developmental Stages ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Class Ii ◽  
Class Iii ◽  
Swarmer Cell ◽  
Sequencing Project

ABSTRACT The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. TheflgBC-fliE genes were identified as a result of the C. crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with ς70 is responsible for regulating transcription of other early flagellar genes in C. crescentus. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, andflgB deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus.

scholarly journals Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter.

1994 ◽  
Vol 14 (10) ◽  
pp. 6607-6615 ◽  
Author(s):  
E Neuman ◽  
E K Flemington ◽  
W R Sellers ◽  
W G Kaelin
Keyword(s):  
Cell Cycle ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Mrna Abundance ◽  
Dna Binding Sites ◽  
Cell Cycle Dependence ◽  
Transcription Factor E2f ◽  
Cycle Dependent

The cell cycle-regulatory transcription factor E2F-1 is regulated by interactions with proteins such as the retinoblastoma gene product and by cell cycle-dependent alterations in E2F-1 mRNA abundance. To better understand this latter phenomenon, we have isolated the human E2F-1 promoter. The human E2F-1 promoter, fused to a luciferase cDNA, gave rise to cell cycle-dependent luciferase activity upon transfection into mammalian cells in a manner which paralleled previously reported changes in E2F-1 mRNA abundance. The E2F-1 promoter contains four potential E2F-binding sites organized as two imperfect palindromes. Gel shift and transactivation studies suggested that these sites can bind to E2F in vitro and in vivo. Mutation of the two E2F palindromes abolished the cell cycle dependence of the E2F-1 promoter. Thus, E2F-1 appears to be regulated at the level of transcription, and this regulation is due, at least in part, to binding of one or more E2F family members to the E2F-1 promoter.

scholarly journals The transcriptional repressor gene Mad3 is a novel target for regulation by E2F1

2003 ◽  
Vol 370 (1) ◽  
pp. 307-313 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
S Phase ◽  
Mobility Shift ◽  
Binding Factor ◽  
Flanking Region ◽  
Novel Target ◽  
Electrophoretic Mobility Shift Assays

Mad family proteins are transcriptional repressors that antagonize the activity of the c-Myc proto-oncogene product. Mad3 is expressed specifically during the S-phase of the cell cycle in both proliferating and differentiating cells, suggesting that its biological function is probably linked to processes that occur during this period. To determine the mechanisms that regulate the cell-cycle-specific transcription of Mad3, we used reporter gene assays in stably transfected fibroblasts. We show that the activation of Mad3 at the G1—S boundary is mediated by a single E2F (E2 promoter binding factor)-binding site within the 5′-flanking region of the gene. Mutation of this element eliminated transcriptional activation at S-phase, suggesting that the positively acting E2F proteins play a role in Mad3 regulation. Using electrophoretic mobility-shift assays and chromatin immunoprecipitation, we show that E2F1 binds to the Mad3 5′-flanking region both in vitro and in vivo. We thus identify Mad3 as a novel transcriptional target of E2F1.

scholarly journals Molecular architecture of the DNA‐binding sites of the P‐loop ATPases MipZ and ParA from Caulobacter crescentus

2019 ◽  
Author(s):  
Yacine Refes ◽  
Binbin He ◽  
Laura Corrales-Guerrero ◽  
Wieland Steinchen ◽  
Gaël Panis ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Electrostatic Interactions ◽  
Caulobacter Crescentus ◽  
Bioinformatic Analysis ◽  
Proper Function ◽  
Molecular Architecture ◽  
Independent Manner ◽  
Dna Binding Sites

ABSTRACTTwo related P-loop ATPases, ParA and MipZ, mediate the spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus. Both of these proteins share the ability to form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization relies on their nucleotide-dependent cycling between a monomeric and a dimeric state, driven by interaction with the chromosome partitioning protein ParB, and on the ability of the dimeric species to associate non-specifically with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to identify the residues mediating the interaction of MipZ with DNA. Our results show that the DNA-binding activity of MipZ relies on a series of positively charged and hydrophobic residues lining both sides of the dimer interface. MipZ thus appears to associate with DNA in a sequence-independent manner through electrostatic interactions with the DNA phosphate backbone. In support of this hypothesis, chromatin immunoprecipitation analyses did not reveal any specific target sites in vivo. When extending our analysis to ParA, we found that the architectures of the MipZ and ParA DNA-binding sites are markedly different, although their relative positions on the dimer surface and their mode of DNA binding are conserved. Importantly, bioinformatic analysis suggests that the same principles apply to other members of the P-loop ATPase family. ParA-like ATPases thus share common mechanistic features, although their modes of action have diverged considerably during the course of evolution.SIGNIFICANCEParA-like P-loop ATPases are involved in a variety of cellular processes in bacteria, including chromosome and plasmid segregation, chemoreceptor and carboxysome positioning, and division site placement. Many members of this large protein family depend on the ability to bind non-specific DNA for proper function. Although previous studies have yielded insights in the DNA-binding properties of some ParA-like ATPases, a comprehensive view of the underlying mechanisms is still lacking. Here, we combine state-of-the-art cell biological, biochemical and biophysical approaches to localize the DNA-binding regions of the ParA-like ATPases MipZ and ParA from Caulobacter crescentus. We show that the two proteins use the same interface and mode of action to associate with DNA, suggesting that the mechanistic basis of DNA binding may be conserved in the ParA-like ATPase family.

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