scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

Enhancement of the in vitro transcription by T7 RNA polymerase of short DNA templates containing oxidative thymine lesions

2005 ◽  
Vol 328 (9) ◽  
pp. 794-801 ◽  
Author(s):  
Valérie Guerniou ◽  
Didier Gasparutto ◽  
Thierry Douki ◽  
Jean Cadet ◽  
Sylvie Sauvaigo
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Dna Templates

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.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

Optimized RNA amplification using T7-RNA-polymerase based in vitro transcription

2004 ◽  
Vol 334 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Pamela R. Moll ◽  
Jutta Duschl ◽  
Klaus Richter
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Amplification

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

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage

1994 ◽  
Vol 14 (6) ◽  
pp. 3596-3603
Author(s):  
H M Dunstan ◽  
L S Young ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Dna Cleavage ◽  
Rna Polymerase Iii ◽  
Low Frequency ◽  
In Vitro Transcription ◽  
Class Iii ◽  
Transcriptional Inhibition ◽  
Transcription In Vitro

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

scholarly journals In vitro transcription through nucleosomes by T7 RNA polymerase.

1992 ◽  
Vol 11 (5) ◽  
pp. 1941-1947 ◽  
Author(s):  
N. Kirov ◽  
I. Tsaneva ◽  
E. Einbinder ◽  
R. Tsanev
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase

scholarly journals T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates

RNA ◽  
1998 ◽  
Vol 4 (10) ◽  
pp. 1313-1317 ◽  
Author(s):  
JEFFREY A. PLEISS ◽  
MARIA L. DERRICK ◽  
OLKE C. UHLENBECK
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase

scholarly journals tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

1994 ◽  
Vol 14 (6) ◽  
pp. 3596-3603 ◽  
Author(s):  
H M Dunstan ◽  
L S Young ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Dna Cleavage ◽  
Rna Polymerase Iii ◽  
Low Frequency ◽  
In Vitro Transcription ◽  
Class Iii ◽  
Transcriptional Inhibition ◽  
Transcription In Vitro

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).

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