scholarly journals Changes in the number of binding sites for ribonucleic acid polymerase in chromatin of WI-38 fibroblasts stimulated to proliferate

1974 ◽  
Vol 141 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Bridget T. Hill ◽  
Renato Baserga
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Template Activity ◽  
E Coli ◽  
Human Diploid Fibroblasts ◽  
Chromatin Template ◽  
Number Of Binding Sites

1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [3H]UTP into RNA in the absence of reinitiation, and incorporation of [γ-32P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.

scholarly journals Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

2005 ◽  
Vol 187 (17) ◽  
pp. 6166-6174 ◽  
Author(s):  
Christopher D. Herring ◽  
Marni Raffaelle ◽  
Timothy E. Allen ◽  
Elenita I. Kanin ◽  
Robert Landick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Reading Frame ◽  
E Coli ◽  
Genome Wide ◽  
Polymerase Binding ◽  
Chip Chip ◽  
Negative Detection

ABSTRACT The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

Evidence for the transcription of physiologically inactive rat-liver nucleolar chromatin by Escherichia coli RNA polymerase

1982 ◽  
Vol 2 (3) ◽  
pp. 155-161 ◽  
Author(s):  
Fu-Li Yu ◽  
Annabella Barrett
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rat Liver ◽  
Dnase I ◽  
Rna Polymerase I ◽  
E Coli ◽  
Chromatin Template ◽  
Nucleolar Chromatin ◽  
Polymerase I ◽  
Active Genes

Rat-liver nucleoli (10–15 pg DNA) were digested with either 0.6 or 3 units of DNase I for various times (up to 1 h). RNA synthesis was then measured in the absence or presence ol 3 units of Escherichia coli RNA polymerase. It was found that the nucleolar chromatin supporting the endogenous engaged RNA polymerase I transcription was compl-etely destroyed in 3 min with either concentration of DNase I. The nucleolar chromatin template transcribed by E. coli RNA polymerase retained 50% of its original capacity even 60 min alter 3 units of DNase I digestion. When hybridization experiments were conducted, it was found that the DNAs derived from both levels of DNase-Idigested nucleoli were incapable of forming hybrids with the labelled nucleolar RNA synthesized by the engaged RNA polymerase I from the untreated nucleoli. Since the engaged RNA polymerase I transcribes only the physiologically active genes of the nucleolar chromatin, and the RNA transcripts represent active gene product, these data suggest that DNase I digestion has completely destroyed the active genes of the nucleolar chromatin, and E. coli RNA polymerase is able to transcribe the inactive nucleolar chromatin template.

scholarly journals Superimposition of TyrR Protein-Mediated Regulation on Osmoresponsive Transcription of Escherichia coli proUIn Vivo

1998 ◽  
Vol 180 (24) ◽  
pp. 6743-6748 ◽  
Author(s):  
J. Gowrishankar ◽  
A. J. Pittard
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Osmotic Regulation ◽  
E Coli ◽  
Regulator Protein ◽  
The Creation

ABSTRACT Osmotic regulation of proU expression in the enterobacteria is achieved, at least in part, by a repression mechanism involving the histone-like nucleoid protein H-NS. By the creation of binding sites for the TyrR regulator protein in the vicinity of the ς70-controlled promoter of proU inEscherichia coli, we were able to demonstrate a superposed TyrR-mediated activation by l-phenylalanine (Phe), as well as repression by l-tyrosine, of proU expression in vivo. Based on the facts that pronounced activation in the presence of Phe was observed even at a low osmolarity and that the affinity of binding of TyrR to its cognate sites on DNA is not affected by Phe, we argue that H-NS-mediated repression of proU at a low osmolarity may not involve a classical silencing mechanism. Our data also suggest the involvement of recruited RNA polymerase in the mechanism of antirepression in E. coli.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

scholarly journals A procedure for the rapid purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase (Short Communication)

1973 ◽  
Vol 133 (1) ◽  
pp. 201-203 ◽  
Author(s):  
Peter Humphries ◽  
David J. McConnell ◽  
Robert L. Gordon
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Short Communication ◽  
Gel Filtration ◽  
High Salt ◽  
Rapid Procedure ◽  
Complete Purification ◽  
Rapid Purification

A rapid procedure involving DNA–cellulose chromatography followed either by sedimentation in a high-salt glycerol gradient or by gel filtration is described for the complete purification of Escherichia coli DNA-dependent RNA polymerase.

scholarly journals Of the Morphogenes that Make a Ring, A Rod and a Sphere in Escherichia Coli

2007 ◽  
Vol 90 (2-3) ◽  
pp. 59-72 ◽  
Author(s):  
Medhatm Khattar ◽  
Issmat I. Kassem ◽  
Ziad W. El-Hajj
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Bacterial Cell ◽  
Bacterial Cell Division ◽  
Antibacterial Compounds ◽  
E Coli ◽  
The Past ◽  
New Knowledge ◽  
Fundamental Process ◽  
Simple Question

In 1993, William Donachie wrote “The success of molecular genetics in the study of bacterial cell division has been so great that we find ourselves, armed with much greater knowledge of detail, confronted once again with the same naive questions that we set to answer in the first place”1. Indeed, attempts to answer the apparently simple question of how a bacterial cell divides have led to a wealth of new knowledge, in particular over the past decade and a half. And while some questions have been answered to a great extent since the early reports of isolation of division mutants of Escherichia coli2,3, some key pieces of the puzzle remain elusive. In addition to it being a fundamental process in bacteria that merits investigation in its own right, studying the process of cell division offers an abundance of new targets for the development of new antibacterial compounds that act directly against key division proteins and other components of the cytoskeleton, which are encoded by the morphogenes of E. coli4. This review aims to present the reader with a snapshot summary of the key players in E. coli morphogenesis with emphasis on cell division and the rod to sphere transition.

scholarly journals High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP

2020 ◽  
Vol 76 (2) ◽  
pp. 94-102 ◽  
Author(s):  
Maria A. Schumacher ◽  
Tomoo Ohashi ◽  
Lauren Corbin ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Cell Division ◽  
High Resolution ◽  
Crystal Structures ◽  
Bacterial Cell ◽  
Model System ◽  
E Coli ◽  
Z Ring ◽  
Main Model

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.

scholarly journals A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

2021 ◽  
Author(s):  
Shirin Ansari ◽  
James C. Walsh ◽  
Amy L. Bottomley ◽  
Iain G. Duggin ◽  
Catherine Burke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Sos Response ◽  
E Coli ◽  
Ring Assembly ◽  
Multiple Alternative ◽  
Z Ring ◽  
Cell Division Inhibitor

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

scholarly journals Fnr-, NarP- and NarL-Dependent Regulation of Transcription Initiation from the Haemophilus influenzae Rd napF (Periplasmic Nitrate Reductase) Promoter in Escherichia coli K-12

2005 ◽  
Vol 187 (20) ◽  
pp. 6928-6935 ◽  
Author(s):  
Valley Stewart ◽  
Peggy J. Bledsoe
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Haemophilus Influenzae ◽  
Control Region ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Class I ◽  
E Coli ◽  
Fnr Protein

ABSTRACT Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions −64.5 and −44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position −60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions −81.5 and −42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.

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