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Related Concept Videos

Bacterial RNA Polymerase00:43

Bacterial RNA Polymerase

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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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Proofreading01:31

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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Updated: May 21, 2025

A Nonsequencing Approach for the Rapid Detection of RNA Editing
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T7 RNA polymerase-guided base editor for accelerated continuous evolution in Bacillus subtilis.

Bin Wang1,2,3, Yaokang Wu1,2,3, Xueqin Lv1,2,3

  • 1School of Biotechnology and Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.

Synthetic and Systems Biotechnology
|May 19, 2025
PubMed
Summary

We developed the BS-MutaT7 system for targeted gene evolution in Bacillus subtilis, enabling rapid genetic diversification through base deaminase-T7 RNA polymerase fusions. This powerful tool accelerates continuous directed evolution for genomic applications.

Keywords:
BS-MutaT7 systemBacillus subtilisContinuous directed evolutionT7 RNA polymerase

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Genetics

Background:

  • Continuous directed evolution accelerates genetic diversification.
  • T7 RNA polymerase (T7 RNAP) fusions are effective for hypermutation.
  • Bacillus subtilis lacked systems for T7 RNAP-guided continuous evolution, limiting genome-targeted diversification.

Purpose of the Study:

  • To develop a novel system for T7 RNAP-guided continuous evolution in Bacillus subtilis.
  • To engineer base deaminase-T7 RNAP fusions for targeted genomic hypermutation.
  • To establish a versatile tool for genome-scale genetic diversification in B. subtilis.

Main Methods:

  • Constructed four libraries using seven deaminases and 14 linkers fused to T7 RNAP.
  • Screened for optimal chimeric mutators based on promoter binding and mutagenesis activity.
  • Evaluated mutation rates, processivity, and application in strain engineering.

Main Results:

  • Identified two optimal mutators, BS-MutaT7^A and BS-MutaT7^C, with significantly increased mutation rates (up to 5.8 × 10^-5 s.p.b.).
  • Demonstrated high processivity, maintaining mutation rates within a 5 kb DNA region.
  • Achieved a 16-fold increase in tigecycline resistance and enhanced β-lactoglobulin expression via codY evolution.

Conclusions:

  • The BS-MutaT7 system enables efficient, targeted, and genome-scale continuous evolution in Bacillus subtilis.
  • BS-MutaT7^C shows superior mutagenic activity for intensive genomic diversification.
  • This system is a powerful tool for accelerating strain improvement and protein expression.