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.

scholarly journals Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro

2018 ◽  
Vol 1861 (8) ◽  
pp. 731-742 ◽  
Author(s):  
Bożena Bruhn-Olszewska ◽  
Vadim Molodtsov ◽  
Michał Sobala ◽  
Maciej Dylewski ◽  
Katsuhiko S. Murakami ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Structure Function ◽  
Binding Sites ◽  
Polymerase Binding

scholarly journals Identification of the miaB Gene, Involved in Methylthiolation of Isopentenylated A37 Derivatives in the tRNA of Salmonella typhimurium andEscherichia coli

1999 ◽  
Vol 181 (23) ◽  
pp. 7256-7265 ◽  
Author(s):  
Birgitta Esberg ◽  
Hon-Chiu Eastwood Leung ◽  
Ho-Ching Tiffany Tsui ◽  
Glenn R. Björk ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Binding Site ◽  
Binding Sites ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Reading Frame ◽  
Conserved Motif ◽  
E Coli ◽  
Weak Similarity

ABSTRACT The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleosideN 6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into thef474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed thatf474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream ofmiaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of themiaB gene, at which the majority (96%) of themiaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms2i(o)6A37 formation.

Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3

Gene ◽  
1983 ◽  
Vol 24 (1) ◽  
pp. 99-113 ◽  
Author(s):  
William L. Wishart ◽  
Machida Chiyoko ◽  
Ohtsubo Hisako ◽  
Ohtsubo Eiichi
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Polymerase Binding

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 RNA polymerase supply and flux through the lac operon in Escherichia coli

2016 ◽  
Vol 371 (1707) ◽  
pp. 20160080 ◽  
Author(s):  
Bandar Sendy ◽  
David J. Lee ◽  
Stephen J. W. Busby ◽  
Jack A. Bryant
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Lac Operon ◽  
Lactose Operon ◽  
E Coli ◽  
Synthetic Promoters ◽  
Promoter Occupancy ◽  
Low Levels ◽  
Polymerase Binding ◽  
Transcript Initiation

Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli . By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase. This article is part of the themed issue ‘The new bacteriology’.

scholarly journals High-resolution, genome-wide mapping of positive supercoiling in chromosomes

2021 ◽  
Author(s):  
Monica S. Guo ◽  
Ryo Kawamura ◽  
Megan Littlehale ◽  
John F. Marko ◽  
Michael T. Laub
Keyword(s):  
High Resolution ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Three Dimensional ◽  
Domain Model ◽  
Bacterial Protein ◽  
E Coli ◽  
Genome Wide ◽  
Powerful Approach ◽  
Positive Supercoiling

AbstractSupercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.

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