Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

2009 ◽  
Vol 113 (46) ◽  
pp. 15399-15408 ◽  
Author(s):  
Abantika Ganguly ◽  
Priya Rajdev ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Thermodynamic Analysis ◽  
Specific Interaction ◽  
Promoter Dna

scholarly journals Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

2003 ◽  
Vol 50 (4) ◽  
pp. 909-920 ◽  
Author(s):  
Iwona K Kolasa ◽  
Tomasz Łoziński ◽  
Kazimierz L Wierzchowski
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Specific Interaction ◽  
Structural Data ◽  
Structural Morphology ◽  
Promoter Dna ◽  
Sigma Subunit ◽  
Kinetics Of

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Transcriptional takeover by σ appropriation: remodelling of the σ 70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1729-1740 ◽  
Author(s):  
Deborah M. Hinton ◽  
Suchira Pande ◽  
Neelowfar Wais ◽  
Xanthia B. Johnson ◽  
Madhavi Vuthoori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Molecular Level ◽  
Bacteriophage T4 ◽  
Good Match ◽  
Promoter Sequences ◽  
Recognition Element ◽  
Upstream Promoter ◽  
Promoter Specificity ◽  
Promoter Dna

Activation of bacteriophage T4 middle promoters, which occurs about 1 min after infection, uses two phage-encoded factors that change the promoter specificity of the host RNA polymerase. These phage factors, the MotA activator and the AsiA co-activator, interact with the σ 70 specificity subunit of Escherichia coli RNA polymerase, which normally contacts the −10 and −35 regions of host promoter DNA. Like host promoters, T4 middle promoters have a good match to the canonical σ 70 DNA element located in the −10 region. However, instead of the σ 70 DNA recognition element in the promoter's −35 region, they have a 9 bp sequence (a MotA box) centred at −30, which is bound by MotA. Recent work has begun to provide information about the MotA/AsiA system at a detailed molecular level. Accumulated evidence suggests that the presence of MotA and AsiA reconfigures protein–DNA contacts in the upstream promoter sequences, without significantly affecting the contacts of σ 70 with the −10 region. This type of activation, which is called ‘σ appropriation’, is fundamentally different from other well-characterized models of prokaryotic activation in which an activator frequently serves to force σ 70 to contact a less than ideal −35 DNA element. This review summarizes the interactions of AsiA and MotA with σ 70, and discusses how these interactions accomplish the switch to T4 middle promoters by inhibiting the typical contacts of the C-terminal region of σ 70, region 4, with the host −35 DNA element and with other subunits of polymerase.

scholarly journals The organization of open complexes between Escherichia coli RNA polymerase and DNA fragments carrying promoters either with or without consensus −35 region sequences

1990 ◽  
Vol 270 (1) ◽  
pp. 141-148 ◽  
Author(s):  
B Chan ◽  
A Spassky ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Point Mutations ◽  
Dna Fragments ◽  
Transcription Start ◽  
Footprint Analysis ◽  
Open Complex Formation ◽  
Promoter Dna

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

scholarly journals Activation by NarL at the Escherichia coli ogt promoter

2020 ◽  
Vol 477 (15) ◽  
pp. 2807-2820
Author(s):  
Patcharawarin Ruanto ◽  
David L. Chismon ◽  
Joanne Hothersall ◽  
Rita E. Godfrey ◽  
David J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Response Regulator ◽  
Direct Interaction ◽  
Α Subunit ◽  
E Coli ◽  
Terminal Domain ◽  
Dna Alkyltransferase ◽  
Promoter Dna ◽  
Transcript Initiation

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

scholarly journals Activation of Bacteriophage Mu momTranscription by C Protein Does Not Require Specific Interaction with the Carboxyl-Terminal Region of the α or ς70Subunit of Escherichia coli RNA Polymerase

1998 ◽  
Vol 180 (12) ◽  
pp. 3257-3259 ◽  
Author(s):  
Weiyong Sun ◽  
Stanley Hattman ◽  
Noboyuki Fujita ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Specific Interaction ◽  
Growth Cycle ◽  
In Vitro Transcription ◽  
Terminal Region ◽  
Bacteriophage Mu ◽  
Phage Gene ◽  
Carboxyl Terminal

ABSTRACT Late in its growth cycle, transcription of the phage Mumom promoter (P mom ) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the α or ς70 subunit of Escherichia coliRNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both α and ς70.

scholarly journals Evidence for a Tyrosine–Adenine Stacking Interaction and for a Short-lived Open Intermediate Subsequent to Initial Binding of Escherichia coli RNA Polymerase to Promoter DNA

2009 ◽  
Vol 385 (2) ◽  
pp. 339-349 ◽  
Author(s):  
Lisa A. Schroeder ◽  
Theodore J. Gries ◽  
Ruth M. Saecker ◽  
M. Thomas Record ◽  
Michael E. Harris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stacking Interaction ◽  
Promoter Dna ◽  
Initial Binding

scholarly journals Structural origins of Escherichia coli RNA polymerase open promoter complex stability

2021 ◽  
Vol 118 (40) ◽  
pp. e2112877118
Author(s):  
Ruth M. Saecker ◽  
James Chen ◽  
Courtney E. Chiu ◽  
Brandon Malone ◽  
Johanna Sotiris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
De Novo ◽  
Promoter Strength ◽  
Transcription Bubble ◽  
Template Strand ◽  
Solution Studies ◽  
Active Site Cleft ◽  
Promoter Dna

The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In Escherichia coli, promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms “open” complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λPR (t1/2 ∼10 h), T7A1 (t1/2 ∼4 min), and a point mutant in λPR (λPR-5C) (t1/2 ∼2 h). Two distinct RPo conformers are populated at λPR, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA–RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand “scrunches” inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.

scholarly journals Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays

2004 ◽  
Vol 186 (20) ◽  
pp. 6938-6943 ◽  
Author(s):  
David C. Grainger ◽  
Timothy W. Overton ◽  
Nikos Reppas ◽  
Joseph T. Wade ◽  
Eiji Tamai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Chromatin Immunoprecipitation ◽  
Positive Control ◽  
Transcription Activator ◽  
Genechip Array ◽  
Genomic Studies ◽  
Affymetrix Genechip Array ◽  
Promoter Dna

ABSTRACT Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

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