In vivo and in vitro activities of point mutants of the bacteriophage T7 RNA polymerase promoter

Biochemistry ◽  
1992 ◽  
Vol 31 (37) ◽  
pp. 9073-9080 ◽  
Author(s):  
Richard A. Ikeda ◽  
G. Sakuntala Warshamana ◽  
Lisa L. Chang
Keyword(s):  
Rna Polymerase ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Rna Polymerase Promoter

scholarly journals Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

2021 ◽  
Author(s):  
Diana Gabriela Calvopina Chavez ◽  
Mikaela Anne Gardner ◽  
Joel S Griffitts
Keyword(s):  
Rna Polymerase ◽  
Molecular Level ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Lower Efficiency ◽  
Transcriptional Termination ◽  
T7 Polymerase

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

scholarly journals In vivo and in vitro phosphorylation of DNA-dependent RNA polymerase of Escherichia coli by bacteriophage-T7-induced protein kinase.

1975 ◽  
Vol 72 (7) ◽  
pp. 2506-2510 ◽  
Author(s):  
W. Zillig ◽  
H. Fujiki ◽  
W. Blum ◽  
D. Janekovic ◽  
M. Schweiger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rna Polymerase ◽  
Bacteriophage T7

scholarly journals Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

2014 ◽  
Author(s):  
Jongmin Kim ◽  
Juan F Quijano ◽  
Enoch Yeung ◽  
Richard M Murray
Keyword(s):  
Rna Polymerase ◽  
Expression System ◽  
Building Blocks ◽  
Logic Circuits ◽  
T7 Rna Polymerase ◽  
Free Expression ◽  
Rna Aptamer ◽  
Cell Free Expression System

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.

Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry

2014 ◽  
Vol 4 (10) ◽  
pp. 1070-1076 ◽  
Author(s):  
Adam J. Meyer ◽  
Jared W. Ellefson ◽  
Andrew D. Ellington
Keyword(s):  
Rna Polymerase ◽  
Directed Evolution ◽  
T7 Rna Polymerase

scholarly journals In vivo and in vitro transcription of T7 early genes by T7 RNA polymerase

FEBS Letters ◽  
1973 ◽  
Vol 33 (3) ◽  
pp. 335-338 ◽  
Author(s):  
J.R. Lillehaug ◽  
D. Helland ◽  
N.O. Sjöberg
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Early Genes

scholarly journals Thymine at —5 Is Crucial for cpc Promoter Activity of Synechocystis sp. Strain PCC 6714

2003 ◽  
Vol 185 (21) ◽  
pp. 6477-6480 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Shoko Fujiwara ◽  
Ryohei Tanigawa ◽  
Kan Tanaka ◽  
Takatsugu Hirokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Pcc 7942 ◽  
Rna Polymerase Promoter ◽  
Synechocystis Sp

ABSTRACT The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp. strain PCC 6714. This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon. Any substitution for T at the −5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp. strain PCC 7942 but not the in vivo activity in Escherichia coli. This suggests that the requirement of −5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination.

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