Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction

Rusakova, E. E.; Tunitskaya, V. L.; Memelova, L. V.; Kochetkova, S. V.; Kostyuk, D. A.; Kochetkov, Sergey N.

doi:10.1016/s0014-5793(98)00058-1

Cited by 7 publications

(4 citation statements)

References 15 publications

(28 reference statements)

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“…The wild‐type T7 RNAP and its mutants were isolated from Escherichia coli strains and purified as described previously [15]. The enzyme activity using synthetic promoter‐containing ONs was determined as described in [16].…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme‐promoter binding for unproductive duplexes was estimated by two methods: (a) measurement of inhibitory effect of the modified duplex on transcription with control (unmodified) duplexes for the wild‐type enzyme ( K i ); (b) direct measurement of promoter binding using sorption on nitrocellulose filters ( K s ) [16]. The constant of covalent binding of mutant enzyme to the modified promoter ( K i irr.)…”

Section: Methodsmentioning

confidence: 99%

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Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

et al. 1999

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Oligonucleotides of a novel type containing 2P-O-L Lribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme. Wild-type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter-containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of proteinnucleic acid interactions.z 1999 Federation of European Biochemical Societies.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

et al. 1999

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“…As a result of this mutational analysis, it was found, for example, that the Y639F mutant has an amazing ability to use both rNTP and dNTP as substrates [ 47 ]. The additional mutation S641A markedly enhanced this property; therefore, the “double” mutant Y639F/S641A was able to synthesize long mixed polynucleotides, where one, two, or three types of rNTPs were replaced by the corresponding dNTPs [ 48 , 49 ]. Moreover, mutant RNA polymerase is used in the development of synthetic nucleotides.…”

Section: Introductionmentioning

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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“…Containing a highly specific promoter recognition site and a nascent RNA-binding domain in its catalytic domain, T7 RNAP works self-sufficiently and drives transcription strongly without transcription factors. 20 , 21 , 22 T7 RNAP exhibits an efficient elongation rate (about 130 nt/s) and displays a relatively high level of template-dependent transcriptional infidelity (2 × 10 −6 error rate). 23 The ability to distinguish between correct and incorrect nucleotides is significant for its high efficiency and fidelity control of transcription.…”

Section: Introductionmentioning

confidence: 99%

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

Liu

Kan

et al. 2020

Molecular Therapy - Nucleic Acids

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Due to highly enzymatic d -stereoselectivity, l -nucleotides ( l -2′-deoxynucleoside 5′-triphosphates [l -dNTPs]) are not natural targets of polymerases. In this study, we synthesized series of l -thymidine ( l -T)-modified DNA strands and evaluated the processivity of nucleotide incorporation for transcription by T7 RNA polymerase (RNAP) with an l -T-containing template. When single l -T was introduced into the transcribed region, transcription proceeded to afford the full-length transcript with different efficiencies. However, introduction of l -T into the non-transcribed region did not exhibit a noticeable change in the transcription efficiency. Surprisingly, when two consecutive or internal l -Ts were introduced into the transcribed region, no transcripts were detected. Compared to natural template, significant lags in NTP incorporation into the template T+4/N and T+7/N (where the number corresponds to the site of l -T position, and + means downstream of the transcribed region) were detected by kinetic analysis. Furthermore, affinity of template T+4/N was almost the same with T/N, whereas affinity of T+7/N was apparently increased. Furthermore, no mismatch opposite to l -T in the template was detected in transcription reactions via gel fidelity analysis. These results demonstrate the effects of chiral l -T in DNA on the efficiency and fidelity of RNA transcription mediated by T7 RNAP, which provides important knowledge about how mirror-image thymidine perturbs the flow of genetic information during RNA transcription and development of diseases caused by gene mutation.

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Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction

Cited by 7 publications

References 15 publications

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

Mapping of T7 RNA polymerase active site with novel reagents – oligonucleotides with reactive dialdehyde groups

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

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

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