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

The Escherichia coli bacteriophage T5 has three temporal classes of genes (pre-early, early, and late). All three classes are transcribed by host RNA polymerase (RNAP) containing the σ70 promoter specificity subunit. Molecular mechanisms responsible for the switching of viral transcription from one class to another remain unknown. Here, we find the product of T5 gene 026 (gpT5.026) in RNAP preparations purified from T5-infected cells and demonstrate in vitro its tight binding to E. coli RNAP. While proteins homologous to gpT5.026 are encoded by all T5-related phages, no similarities to proteins with known functions can be detected. GpT5.026 binds to two regions of the RNAP β subunit and moderately inhibits RNAP interaction with the discriminator region of σ70-dependent promoters. A T5 mutant with disrupted gene 026 is viable, but the host cell lysis phase is prolongated and fewer virus particles are produced. During the mutant phage infection, the number of early transcripts increases, whereas the number of late transcripts decreases. We propose that gpT5.026 is part of the regulatory cascade that orchestrates a switch from early to late bacteriophage T5 transcription.

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

Gene ◽

10.1016/0378-1119(83)90135-x ◽

1983 ◽

Vol 24 (1) ◽

pp. 99-113 ◽

Cited By ~ 17

Author(s):

William L. Wishart ◽

Machida Chiyoko ◽

Ohtsubo Hisako ◽

Ohtsubo Eiichi

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Binding Sites ◽

Transcription Initiation ◽

Polymerase Binding

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Mapping of Escherichia coli RNA polymerase binding sites on 2-μm DNA from Saccharomyces cerevisiae

Plasmid ◽

10.1016/0147-619x(79)90024-6 ◽

1979 ◽

Vol 2 (3) ◽

pp. 403-416 ◽

Cited By ~ 3

Author(s):

Hans-Dieter Royer ◽

Cornelis P. Hollenberg

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Binding Sites ◽

Polymerase Binding

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

Journal of Bacteriology ◽

10.1128/jb.187.17.6166-6174.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 6166-6174 ◽

Cited By ~ 83

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.

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A comparison of the phage T4 gene 32 protein and Escherichia coli RNA polymerase binding sites on hamster papovavirus DNA

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(82)90015-x ◽

1982 ◽

Vol 696 (1) ◽

pp. 102-106 ◽

Cited By ~ 2

Author(s):

Frank Vogel ◽

Siegfried Scherneck

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Binding Sites ◽

Phage T4 ◽

Polymerase Binding ◽

Gene 32 ◽

T4 Gene 32 Protein

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

Journal of Cell Science ◽

10.1242/jcs.17.3.287 ◽

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.

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