scholarly journals Footprinting analysis of the interactions between E. coli RNA polymerase and promoter DNA that produce very long abortive transcripts (VLATs)

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Mya Thandar ◽  
Ahri Lee ◽  
Lilian M Hsu
Keyword(s):  
Rna Polymerase ◽  
E Coli ◽  
Promoter Dna

scholarly journals E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation and dynamics

2019 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y. Chen ◽  
Elizabeth A. Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
E Coli ◽  
Bacterial Proteins ◽  
Cryo Electron Microscopy ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

AbstractTraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp. How TraR and its homologs regulate transcription is unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis of RNAP conformational heterogeneity revealed TraR-induced changes in RNAP dynamics. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

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 E. coli TraR allosterically regulates transcription initiation by altering RNA polymerase conformation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
James Chen ◽  
Saumya Gopalkrishnan ◽  
Courtney Chiu ◽  
Albert Y Chen ◽  
Elizabeth A Campbell ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Structural Basis ◽  
E Coli ◽  
Bacterial Proteins ◽  
Promoter Complex ◽  
Promoter Dna ◽  
Induced Changes

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters.

scholarly journals Fluorescence-Detected Conformational Changes in Duplex DNA in Open Complex Formation by E. coli RNA Polymerase: Upstream Wrapping and Downstream Bending Precede Clamp Opening and Insertion of the Downstream Duplex

2020 ◽  
Author(s):  
Raashi Sreenivasan ◽  
Irina A Shkel ◽  
Munish Chhabra ◽  
Amanda Drennan ◽  
Sara Heitkamp ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Large Scale ◽  
Dna Bending ◽  
Duplex Dna ◽  
E Coli ◽  
Fret Efficiency ◽  
Two Phases ◽  
Promoter Dna ◽  
Transcription Activators

FRET (fluorescence energy transfer) between far-upstream (-100) and downstream (+14) cyanine dyes showed extensive bending/wrapping of λPR promoter DNA on E. coli RNA polymerase (RNAP) in closed and open complexes (CC, OC). Here we determine the kinetics and mechanism of DNA bending/wrapping by FRET and of formation of RNAP contacts with -100 and +14 DNA by single-dye fluorescence enhancements (PIFE). FRET/PIFE kinetics exhibit two phases: rapidly-reversible steps forming a CC ensemble ({CC} of four intermediates (initial (RPc), early (I1E), mid- (I1M), late (I1L)), followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET/PIFE together reveal large-scale bending/wrapping of upstream and downstream DNA as RPc advances to I1E, reducing -100/+14 distance to 75 Å and making RNAP-DNA contacts at -100 and +14. We propose that far-upstream DNA wraps on the upper β′-clamp while downstream DNA contacts the top of the β-pincer in I1E. Converting I1E to I1M (~1s time-scale) reduces FRET efficiency with little change in -100/+14 PIFE, interpreted as clamp-opening that moves far-upstream DNA (on β′) away from downstream DNA (on β) to increase the -100/+14 distance by 14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp-closing to reduce the -100/+14 distance by 21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L converts to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream-binding, DNA-bending transcription activators are discussed.

scholarly journals Bending and Wrapping of Upstream Promoter DNA on E. coli RNA Polymerase Facilitates Open Complex Formation in Transcription Initiation; A Fluorescence (FRET, PIFE) Study

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Christina Lynne McNerney ◽  
Katelyn Callies ◽  
Clare Kai Cimperman ◽  
Priya Chittur ◽  
Raashi Sreenivasan ◽  
...  
Keyword(s):  
Complex Formation ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
E Coli ◽  
Upstream Promoter ◽  
Open Complex Formation ◽  
Promoter Dna

scholarly journals Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Juncao Xu ◽  
Kaijie Cui ◽  
Liqiang Shen ◽  
Jing Shi ◽  
Lingting Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Activation ◽  
Initiation Factor ◽  
Bacterial Cells ◽  
E Coli ◽  
Exchange Mass Spectrometry ◽  
Transcription Initiation Factor ◽  
Hydrogen Deuterium ◽  
Promoter Dna

σS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNA polymerase (σS-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σS (σS2) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σS2 to promote the assembly of the σS-RNAP holoenzyme and also to facilitate formation of an RNA polymerase–promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

scholarly journals Base-Specific Recognition of the Nontemplate Strand of Promoter DNA by E. coli RNA Polymerase

Cell ◽  
1996 ◽  
Vol 86 (3) ◽  
pp. 495-501 ◽  
Author(s):  
Jeffrey W Roberts ◽  
Christine W Roberts
Keyword(s):  
Rna Polymerase ◽  
E Coli ◽  
Specific Recognition ◽  
Promoter Dna

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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