scholarly journals Direct Observation of Abortive Initiation and Promoter Escape within Single Immobilized Transcription Complexes

2006 ◽  
Vol 90 (4) ◽  
pp. 1419-1431 ◽  
Author(s):  
Emmanuel Margeat ◽  
Achillefs N. Kapanidis ◽  
Philip Tinnefeld ◽  
You Wang ◽  
Jayanta Mukhopadhyay ◽  
...  
Keyword(s):  
Direct Observation ◽  
Promoter Escape ◽  
Transcription Complexes ◽  
Abortive Initiation

scholarly journals Translocation after Synthesis of a Four-Nucleotide RNA Commits RNA Polymerase II to Promoter Escape

2002 ◽  
Vol 22 (3) ◽  
pp. 762-773 ◽  
Author(s):  
Jennifer F. Kugel ◽  
James A. Goodrich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Sequence Specificity ◽  
Promoter Escape ◽  
General Transcription Factors ◽  
Dna Elements ◽  
Promoter Dna ◽  
Transcription Complexes

ABSTRACT Transcription is a complex process, the regulation of which is crucial for cellular and organismic growth and development. Deciphering the molecular mechanisms that define transcription is essential to understanding the regulation of RNA synthesis. Here we describe the molecular mechanism of escape commitment, a critical step in early RNA polymerase II transcription. During escape commitment ternary transcribing complexes become stable and committed to proceeding forward through promoter escape and the remainder of the transcription reaction. We found that the point in the transcription reaction at which escape commitment occurs depends on the length of the transcript RNA (4 nucleotides [nt]) as opposed to the position of the active site of the polymerase with respect to promoter DNA elements. We found that single-stranded nucleic acids can inhibit escape commitment, and we identified oligonucleotides that are potent inhibitors of this specific step. These inhibitors bind RNA polymerase II with low nanomolar affinity and sequence specificity, and they block both promoter-dependent and promoter-independent transcription, the latter occurring in the absence of general transcription factors. We demonstrate that escape commitment involves translocation of the RNA polymerase II active site between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4-nt RNA to render complexes escape committed.

scholarly journals TFIIH Operates through an Expanded Proximal Promoter To Fine-Tune c-myc Expression

2005 ◽  
Vol 25 (1) ◽  
pp. 147-161 ◽  
Author(s):  
Achim Weber ◽  
Juhong Liu ◽  
Irene Collins ◽  
David Levens
Keyword(s):  
Intrinsic Noise ◽  
Complementation Group ◽  
Proximal Promoter ◽  
Promoter Escape ◽  
General Transcription Factor ◽  
Continuous Stream ◽  
Transcription Complexes ◽  
Group B ◽  
Cell Variation ◽  
Transcriptional Output

ABSTRACT A continuous stream of activating and repressing signals is processed by the transcription complex paused at the promoter of the c-myc proto-oncogene. The general transcription factor IIH (TFIIH) is held at promoters prior to promoter escape and so is well situated to channel the input of activators and repressors to modulate c-myc expression. We have compared cells expressing only a mutated p89 (xeroderma pigmentosum complementation group B [XPB]), the largest TFIIH subunit, with the same cells functionally complemented with the wild-type protein (XPB/wt-p89). Here, we show structural, compositional, and functional differences in transcription complexes between XPB and XPB/wt-89 cells at the native c-myc promoter. Remarkably, although the mean levels of c-Myc are only modestly elevated in XPB compared to those in XPB/wt-p89 cells, the range of expression and the cell-to-cell variation of c-Myc are markedly increased. Our modeling indicates that the data can be explained if TFIIH integrates inputs from multiple signals, regulating transcription at multiple kinetically equivalent steps between initiation and promoter escape. This helps to suppress the intrinsic noise of transcription and to ensure the steady transcriptional output of c-myc necessary for cellular homeostasis.

Interplay between σ region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase

2017 ◽  
Vol 474 (24) ◽  
pp. 4053-4064 ◽  
Author(s):  
Ivan Petushkov ◽  
Daria Esyunina ◽  
Vladimir Mekler ◽  
Konstantin Severinov ◽  
Danil Pupov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Molecular Beacon ◽  
Secondary Channel ◽  
Enzyme Active Site ◽  
Promoter Escape ◽  
Bacterial Rna ◽  
Transcription Complexes ◽  
Kinetics Of ◽  
Rnap Binding

In bacterial RNA polymerase (RNAP), conserved region 3.2 of the σ subunit was proposed to contribute to promoter escape by interacting with the 5′-end of nascent RNA, thus facilitating σ dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled σ70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in σ region 3.2 decrease the rate of promoter escape and lead to accumulation of inactive complexes during transcription initiation. Secondary channel factors differentially regulate this process depending on the promoter and mutations in σ region 3.2. GreA generally increase the rate of promoter escape; DksA also stimulates promoter escape on certain templates, while GreB either stimulates or inhibits this process depending on the template. When observed, the stimulation of promoter escape correlates with the accumulation of stressed transcription complexes with scrunched DNA, while changes in the RNA 5′-end structure modulate promoter clearance. Thus, the initiation-to-elongation transition is controlled by a complex interplay between RNAP-binding protein factors and the growing RNA chain.

scholarly journals Strong Natural Pausing by RNA Polymerase II within 10 Bases of Transcription Start May Result in Repeated Slippage and Reextension of the Nascent RNA

2002 ◽  
Vol 22 (1) ◽  
pp. 30-40 ◽  
Author(s):  
Mahadeb Pal ◽  
Donal S. Luse
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Early Stage ◽  
Transcription Start ◽  
Promoter Escape ◽  
Transcript Length ◽  
Template Sequence ◽  
Transcription Complexes ◽  
Transcript Initiation

ABSTRACT We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 μM) levels of nucleoside triphosphates. Strong pauses with half-lives of >30 s were observed at +7, +18/19, and about +25 on the template used in these experiments. We show that the strong pause at +7, after the synthesis of 5′-ACUCUCU, leads to repeated cycles of upstream slippage of the RNA-DNA hybrid followed by re-pairing with the DNA and continued RNA synthesis. The resulting transcripts are 2, 4, and 6 bases longer than predicted by the template sequence. Slippage is efficient when transcription is primed with the +1/+2 (ApC) dinucleotide, and it occurs at even higher levels with the +2/+3 primer (CpU). Slippage can occur at high levels with ATP initiation, but priming with CpA (−1/+1) supports very little slippage. This latter result is not simply an effect of transcript length at the point of pausing. Slippage can also occur with a second template on which the polymerase can be paused after synthesizing ACUCU. Slippage is not reduced by an ATP analog that blocks promoter escape, but it is inhibited by substitution of 5Br-U for U in the RNA. Our results reveal an unexpected flexibility of RNA polymerase II ternary complexes during the very early stage of transcription, and they suggest that initiation at different locations within the same promoter gives rise to transcription complexes with different properties.

Direct Observation of Heat Exchanger Deposits by Cross Sectioning

1967 ◽  
Vol 25 ◽  
pp. 364-365
Author(s):  
R. W. Anderson ◽  
D. L. Senecal
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Heat Exchanger ◽  
Direct Observation ◽  
Cross Sections ◽  
Preparation Method ◽  
Specimen Preparation ◽  
Flowing Water ◽  
Transmission Electron ◽  
Deposit Layer

A problem was presented to observe the packing densities of deposits of sub-micron corrosion product particles. The deposits were 5-100 mils thick and had formed on the inside surfaces of 3/8 inch diameter Zircaloy-2 heat exchanger tubes. The particles were iron oxides deposited from flowing water and consequently were only weakly bonded. Particular care was required during handling to preserve the original formations of the deposits. The specimen preparation method described below allowed direct observation of cross sections of the deposit layers by transmission electron microscopy.The specimens were short sections of the tubes (about 3 inches long) that were carefully cut from the systems. The insides of the tube sections were first coated with a thin layer of a fluid epoxy resin by dipping. This coating served to impregnate the deposit layer as well as to protect the layer if subsequent handling were required.

Application of a Simple Cold Stub to the Direct Observation of Frozen Hydrated Sand

1973 ◽  
Vol 31 ◽  
pp. 212-213
Author(s):  
John M. Wehrung ◽  
Richard J. Harniman
Keyword(s):  
United States ◽  
Surface Water ◽  
Direct Observation ◽  
Controlled Study ◽  
Clay Particles ◽  
Organic Debris ◽  
Rock Formations ◽  
Water Tables ◽  
Permeable Rock ◽  
Natural Means

Water tables in aquifer regions of the southwest United States are dropping off at a rate which is greater than can be replaced by natural means. It is estimated that by 1985 wells will run dry in this region unless adequate artificial recharging can be accomplished. Recharging with surface water is limited by the plugging of permeable rock formations underground by clay particles and organic debris.A controlled study was initiated in which sand grains were used as the rock formation and water with known clay concentrations as the recharge media. The plugging mechanism was investigated by direct observation in the SEM of frozen hydrated sand samples from selected depths.

Direct Observation of the Diffusionless β → β + ω Transformation in Titanium Alloys

1971 ◽  
Vol 29 ◽  
pp. 122-123
Author(s):  
N. E. Paton ◽  
D. de Fontaine ◽  
J. C. Williams
Keyword(s):  
Electron Microscope ◽  
Titanium Alloys ◽  
Direct Observation ◽  
Dark Field ◽  
X Ray Diffraction ◽  
Resistivity Measurements ◽  
Selected Area Electron Diffraction ◽  
Ti Alloys ◽  
Thin Foils ◽  
Field Device

The electron microscope has been used to study the diffusionless β → β + ω transformation occurring in certain titanium alloys at low temperatures. Evidence for such a transformation was obtained by Cometto et al by means of x-ray diffraction and resistivity measurements on a Ti-Nb alloy. The present work shows that this type of transformation can occur in several Ti alloys of suitable composition, and some of the details of the transformation are elucidated by means of direct observation in the electron microscope.Thin foils were examined in a Philips EM-300 electron microscope equipped with a uniaxial tilt, liquid nitrogen cooled, cold stage and a high resolution dark field device. Selected area electron diffraction was used to identify the phases present and the ω-phase was imaged in dark field by using a (101)ω reflection. Alloys were water quenched from 950°C, thinned, and mounted between copper grids to minimize temperature gradients in the foil.

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