Structure of Sigma-Dependent Binding Sites of E. Coli RNA Polymerase to Phages λ, T5 and T7 DNA’s

1973 ◽  
pp. 301-307
Author(s):  
Philippe Jeanteur ◽  
Jean-Yves Le Talaër
Keyword(s):  
Rna Polymerase ◽  
Binding Sites ◽  
E Coli

scholarly journals Binding sites of E. coli RNA polymerase on T7 DNA as determined by electron microscopy

FEBS Letters ◽  
1974 ◽  
Vol 45 (1-2) ◽  
pp. 64-67 ◽  
Author(s):  
R. Portmann ◽  
J.M. Sogo ◽  
Th. Koller ◽  
W. Zillig
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Sites ◽  
E Coli

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 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.

Electron-microscopic mapping of AT-rich regions and of E. coli RNA polymerase-binding sites on the circular kinetoplast DNA of Trypanosoma cruzi

1975 ◽  
Vol 17 (3) ◽  
pp. 287-306
Author(s):  
C. Brack ◽  
E. Delain
Keyword(s):  
Dna Replication ◽  
Trypanosoma Cruzi ◽  
Rna Polymerase ◽  
Binding Sites ◽  
Specific Binding ◽  
Kinetoplast Dna ◽  
Electron Microscopic ◽  
E Coli ◽  
Polymerase Binding

Partial alkaline denaturation of the circular kinetoplast DNA (kDNA) of Trypanosoma cruzi has shown the existence of 4 small, well-defined AT-rich regions with an average size of about 200 base pairs. They are almost equally distributed, separated by approximately 90 degrees on the circular molecule. All minicircles, whether free or linked in networks, have the same denaturation pattern and, therefore, seem to contain the same information. The long linear molecules present in low amounts in the kDNA samples do not show the same denaturation pattern. Partial denaturation of molecules in larger associations indicates that the circular units may be linked to each other by one strand only. kDNA can be transcribed in vitro by the RNA polymerase of E. coli. RNA polymerase-kDNA complexes have been studied in the electron microscope. By spreading the DNA-protein complexes by adhesion to positively charged carbon films and dark-field observation, it was possible to show the existence of 4 specific binding sites of the E. coli RNA polymerase on the kDNA circles. Comparing the position of the polymerase-binding sites and the AT-rich melted zones, it is suggested that a correlation exists between the two. As had been shown in earlier work, the replication of circular kDNA can be blocked by treating the trypanosomes with the trypanocidal drug Berenil. The comparison of the relative position of the Berenil-blocked replication forks with the position of the 4 denaturation loops shows that the DNA replication is stopped at these AT-rich regions. Since there is evidence that Berenil binds preferentially to AT-rich DNA and seems to be involved in inhibition of DNA replication, the following hypothetical model can be proposed. The replication of the circular kDNA molecules is discontinuous and involves the synthesis of RNA primers; when Berenil is bound to the AT-rich regions, synthesis of new RNA primers is inhibited and replication is blocked at these points, leading to the accumulation of replicating intermediates with defined branch lengths.

scholarly journals Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Llorenç Fernández-Coll ◽  
Katarzyna Potrykus ◽  
Michael Cashel ◽  
Carlos Balsalobre
Keyword(s):  
Rna Polymerase ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Secondary Channel ◽  
Reporter System ◽  
E Coli ◽  
Functional Studies ◽  
The Family ◽  
Pause Site

Abstract There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them. GreA is characterised to rescue stalled RNAP complexes due to its antipause activity, but also it is involved in transcription fidelity and proofreading. Here, overexpression of GreA is noted to be lethal independent of its antipause activity. A library of random GreA variants has been used to isolate lethality suppressors to assess important residues for GreA functionality and its interaction with the RNA polymerase. Some mutant defects are inferred to be associated with altered binding competition with DksA, while other variants seem to have antipause activity defects that cannot reverse a GreA-sensitive pause site in a fliC::lacZ reporter system. Surprisingly, apparent binding and cleavage defects are found scattered throughout both the coiled-coil and globular domains. Thus, the coiled-coil of GreA is not just a measuring stick ensuring placement of acidic residues precisely at the catalytic centre but also seems to have binding functions. These lethality suppressor mutants may provide valuable tools for future structural and functional studies.

Binding Sites of E. coli DNA-dependent RNA polymerase on spinach chloroplast DNA

1981 ◽  
Vol 4 (1) ◽  
pp. 37-46 ◽  
Author(s):  
Michael Zech ◽  
Martin R. Hartley ◽  
Hans J. Bohnert
Keyword(s):  
Rna Polymerase ◽  
Chloroplast Dna ◽  
Binding Sites ◽  
Spinach Chloroplast ◽  
E Coli

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.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

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