scholarly journals G Protein–Coupled Estrogen Receptor Production Using an Escherichia coli Cell‐Free Expression System

2019 ◽  
Vol 97 (1) ◽  
Author(s):  
Samson A. Souza ◽  
Dane T. Kurohara ◽  
Chester L. Dabalos ◽  
Ho Leung Ng
Keyword(s):  
Escherichia Coli ◽  
Estrogen Receptor ◽  
G Protein ◽  
Expression System ◽  
Coli Cell ◽  
Free Expression ◽  
G Protein Coupled ◽  
Cell Free Expression System

Mechanism of estrogen receptor-dependent transcription in a cell-free system

1990 ◽  
Vol 10 (12) ◽  
pp. 6607-6612
Author(s):  
J F Elliston ◽  
S E Fawell ◽  
L Klein-Hitpass ◽  
S Y Tsai ◽  
M J Tsai ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Expression System ◽  
Steroid Receptor ◽  
Free Expression ◽  
Independent Manner ◽  
Free System ◽  
A Cell ◽  
Regulated Gene Expression ◽  
Cell Free Expression System

RNA synthesis was stimulated directly in a cell-free expression system by crude preparations of recombinant mouse estrogen receptor (ER). Receptor-stimulated transcription required the presence of estrogen response elements (EREs) in the test template and could be specifically inhibited by addition of competitor oligonucleotides containing EREs. Moreover, polyclonal antibodies directed against the DNA-binding region of ER inhibited ER-dependent transcription. In our cell-free expression system, hormone-free ER induced transcription in a hormone-independent manner. Evidence is presented suggesting that ER acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II. These observations lend support to our current understanding of the mechanism of steroid receptor-regulated gene expression and suggest strong conservation of function among members of the steroid receptor superfamily.

scholarly journals Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

10.3791/50762 ◽  
2013 ◽  
Author(s):  
Zachary Z. Sun ◽  
Clarmyra A. Hayes ◽  
Jonghyeon Shin ◽  
Filippo Caschera ◽  
Richard M. Murray ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Expression System ◽  
Free Expression ◽  
Cell Free Expression System

scholarly journals A Methylation-Directed, Synthetic Pap Switch Based on Self-Complementary Regulatory DNA in an All-E. Coli Cell-Free Expression System

2020 ◽  
Author(s):  
Emanuel Worst ◽  
oemer Kurt ◽  
Marc Finkler ◽  
Marc Schenkelberger ◽  
Vincent Noireaux ◽  
...  
Keyword(s):  
Expression System ◽  
Regulatory Region ◽  
Phase Variation ◽  
Coli Strain ◽  
Coli Cell ◽  
Free Expression ◽  
E Coli ◽  
Regulatory Dna ◽  
Cell Free Expression System

<p>Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable 'ON phase' where the corresponding pap genes are expressed and a stable 'OFF phase' where their transcription is repressed. Hereditary, alternate DNA methylation of only two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study the function of pap regulatory region based on a specially designed, synthetic construct flanked by two reporter genes encoding fluorescent proteins for simple readout. Based on our observations we suggest that Lrp and the conformation of the self-complementary regulatory DNA play a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for engineering stable, hereditary and strong expression control based on methylation.</p>

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