Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

AbstractPromoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10−20-bp distance from many promoters. The pauses in the 10−15-bp register of the promoter are dictated by the canonical −10 element, 6−7 nt spacer and “YR+1Y” motif centered at the transcription start site. The promoters for the pauses in the 16−20-bp register contain an additional −10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

Download Full-text

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

10.1101/2020.10.25.354225 ◽

2020 ◽

Author(s):

Zhe Sun ◽

Alexander Yakhnin ◽

Peter C. FitzGerald ◽

Carl E. Mclntosh ◽

Mikhail Kashlev

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Environmental Cues ◽

Start Site ◽

Transcription Start ◽

E Coli ◽

Eukaryotic Genes ◽

Genome Wide ◽

Transcript Cleavage

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

Download Full-text

The native structure of the eukaryotic ribosome

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075890 ◽

1983 ◽

Vol 41 ◽

pp. 432-433

Author(s):

R.A. Milligan ◽

P.N.T. Unwin

Keyword(s):

Ribosomal Proteins ◽

Crystal Form ◽

Native Structure ◽

X Ray Diffraction ◽

Eukaryotic Ribosome ◽

Vitro Analysis ◽

In Vitro Analysis ◽

Resolution Structure ◽

Stable Unit

A detailed understanding of the mechanism of protein synthesis will ultimately depend on knowledge of the native structure of the ribosome. Towards this end we have investigated the low resolution structure of the eukaryotic ribosome embedded in frozen buffer, making use of a system in which the ribosomes crystallize naturally.The ribosomes in the cells of early chicken embryos form crystalline arrays when the embryos are cooled at 4°C. We have developed methods to isolate the stable unit of these arrays, the ribosome tetramer, and have determined conditions for the growth of two-dimensional crystals in vitro, Analysis of the proteins in the crystals by 2-D gel electrophoresis demonstrates the presence of all ribosomal proteins normally found in polysomes. There are in addition, four proteins which may facilitate crystallization. The crystals are built from two oppositely facing P4 layers and the predominant crystal form, accounting for >80% of the crystals, has the tetragonal space group P4212, X-ray diffraction of crystal pellets demonstrates that crystalline order extends to ~ 60Å.

Download Full-text

1163: Radial Dilation of Ureteral Balloons: A Comparative in-Vitro Analysis

The Journal of Urology ◽

10.1016/s0022-5347(18)35308-4 ◽

2005 ◽

Vol 173 (4S) ◽

pp. 315-316

Author(s):

Kari Hendlin ◽

Brynn Lund ◽

Manoj Monga

Keyword(s):

Vitro Analysis ◽

In Vitro Analysis

Download Full-text

An In Vitro Analysis of Ligament Reconstruction or Extension Osteotomy on Trapeziometacarpal Joint Stability and Contact Area

Yearbook of Hand and Upper Limb Surgery ◽

10.1016/s1551-7977(08)70357-1 ◽

2007 ◽

Vol 2007 ◽

pp. 214-215 ◽

Cited By ~ 1

Author(s):

B. Wilhelmi

Keyword(s):

Contact Area ◽

Ligament Reconstruction ◽

Joint Stability ◽

Trapeziometacarpal Joint ◽

Vitro Analysis ◽

In Vitro Analysis

Download Full-text

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614605 ◽

1999 ◽

Vol 81 (06) ◽

pp. 951-956 ◽

Cited By ~ 59

Author(s):

J. Corral ◽

R. González-Conejero ◽

J. Rivera ◽

F. Ortuño ◽

P. Aparicio ◽

...

Keyword(s):

Venous Thrombosis ◽

Cell Types ◽

Receptor Expression ◽

Case Control Studies ◽

Platelet Surface ◽

Vitro Analysis ◽

And Function ◽

In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

Download Full-text