Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase

Boulain, Jean‐Claude; Dassa, Janie; Mesta, Laurent; Savatier, Alexandra; Costa, Narciso; Müller, Bruno; L’Hostis, Guillaume; Stura, E.A.; Troesch, Alain; Ducancel, Frédéric

doi:10.1093/protein/gzt040

Cited by 11 publications

(17 citation statements)

References 37 publications

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“…The most common phage RNAP system is the T7 RNAP, a single subunit RNAP that recognizes a single DNA motif between 17 and 22 bp long. The T7 RNA polymerase has appealing properties for synthetic biological applications: it can produce very high transcription rates ( 31 , 32 ) useful for example in amplification steps or high-gain feedback circuits ( 33 ); its compact single-unit nature makes it relatively easy to express; and it has been functionally expressed in a variety of contexts including both prokaryotes and eukaryotes ( 34–37 ).…”

Section: Introductionmentioning

confidence: 99%

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Programmable T7-based synthetic transcription factors

Hussey

McMillen

2018

Nucleic Acids Research

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Despite recent progress on synthetic transcription factor generation in eukaryotes, there remains a need for high-activity bacterial versions of these systems. In synthetic biology applications, it is useful for transcription factors to have two key features: they should be orthogonal (influencing only their own targets, with minimal off-target effects), and programmable (able to be directed to a wide range of user-specified transcriptional start sites). The RNA polymerase of the bacteriophage T7 has a number of appealing properties for synthetic biological designs: it can produce high transcription rates; it is a compact, single-subunit polymerase that has been functionally expressed in a variety of organisms; and its viral origin reduces the connection between its activity and that of its host's transcriptional machinery. We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein–protein interactions, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli. To our knowledge this is the first exogenous, programmable activator system in bacteria.

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Section: Introductionmentioning

confidence: 99%

“…Numerous successful efforts have been made to engineer the T7 RNAP to recognize different promoter sequences ( 38–42 ) as well as to generally enhance its activity ( 31 , 32 ). Further, T7 RNAP has been split to generate inducible ( 43 , 44 ) and logic gated operations ( 45–47 ).…”

Section: Introductionmentioning

confidence: 99%

Programmable T7-based synthetic transcription factors

Hussey

McMillen

2018

Nucleic Acids Research

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“…Most of the other tested mutant forms (P266L, N433Q, G542V, R627S, S633P, H772A) decreased their activity in the coupled 2 system to 10%–30%. For all these mutants, increased thermal stability was demonstrated earlier [ 50 , 54 , 55 , 56 ]. Furthermore, for G542V the violation of interaction with 2′-OH of ribonucleotides was demonstrated [ 50 ] and for R627S, the violation of specificity expressed in the incorporation of ddNTP to RNA was shown [ 54 , 55 ].…”

Section: Resultsmentioning

confidence: 64%

“…Furthermore, for G542V the violation of interaction with 2′-OH of ribonucleotides was demonstrated [ 50 ] and for R627S, the violation of specificity expressed in the incorporation of ddNTP to RNA was shown [ 54 , 55 ]. Additionally, for the mutants S633P and H772A, relative activities in vitro by fluorescent beacon were determined, which were 56% and 70%, respectively [ 56 ]. In the coupled 2 system, we observed the lower activities of these mutants by 30% and 10%, respectively; however, mutants with replacements in position F849 retained more than half of the activity of the T7wt RNAP in the coupled 2 system—F849I, F849A, and F849Y.…”

Section: Resultsmentioning

confidence: 99%

Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides

Егорова

Shuvalova

Mukba

et al. 2021

IJMS

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Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity.

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“…Isothermal and transcription-based amplification of nucleic acids can be performed using T7RP, which is responsible for strong transcription from P T 7 (Niemz et al, 2011). Because amplification performance is potentially improved when conducted at higher temperatures, thermostable T7RP variants are of biotechnological importance (Boulain et al, 2013). This study was originally designed to generate mutant genes for thermostable T7RP variants in G. kaustophilus MK534 T 7RP .…”

Section: Discussionmentioning

confidence: 99%

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

et al. 2021

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Geobacillus kaustophilus HTA426 is a thermophilic bacterium whose genome harbors numerous insertion sequences (IS). This study was initially conducted to generate mutant genes for thermostable T7 RNA polymerase in G. kaustophilus; however, relevant experiments unexpectedly identified that the organism transposed multiple IS elements and produced derivative cells that expressed a silent gene via transposition. The transposed elements were diverse and included members of the IS4, IS701, IS1634, and ISLre2 families. The transposition was relatively active at elevated temperatures and generated 4–9 bp of direct repeats at insertion sites. Transposition was more frequent in proliferative cells than in stationary cells but was comparable between both cells when sigX, which encodes an extra-cytoplasmic function sigma factor, was forcibly expressed. Southern blot analysis indicated that IS transposition occurred under growth inhibitory conditions by diverse stressors; however, IS transposition was not detected in cells that were cultured under growth non-inhibitory conditions. These observations suggest that G. kaustophilus enhances IS transposition via sigX-dependent stress responses when proliferative cells were prevented from active propagation. Considering Geobacillus spp. are highly adaptive bacteria that are remarkably distributed in diverse niches, it is possible that these organisms employ IS transposition for environmental adaptation via genetic diversification. Thus, this study provides new insights into adaptation strategies of Geobacillus spp. along with implications for strong codependence between mobile genetic elements and highly adaptive bacteria for stable persistence and evolutionary diversification, respectively. This is also the first report to reveal active IS elements at elevated temperatures in thermophiles and to suggest a sigma factor that governs IS transposition.

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Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase

Cited by 11 publications

References 37 publications

Programmable T7-based synthetic transcription factors

Programmable T7-based synthetic transcription factors

Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

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