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Updated: Jun 28, 2026
09:26
DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Published on: December 29, 2021
Recombinant bacterial RNA polymerase: preparation and applications
Konstantin Kuznedelov1, Konstantin Severinov
1Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA. kuznedelo@waksman.rutgers.edu
Methods (San Diego, Calif.)
|October 25, 2008
Summary
Researchers developed two key methods for producing bacterial DNA-dependent RNA polymerase. These techniques enable the creation of specific in vitro transcription systems for studying gene regulation in diverse bacterial species.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bacterial DNA-dependent RNA polymerase is crucial for gene transcription.
- Understanding species-specific transcription regulation requires tailored in vitro systems.
- Availability of purified RNA polymerase is essential for these systems.
Purpose of the Study:
- To describe two effective strategies for preparing recombinant bacterial RNA polymerase.
- To enable the development of custom in vitro transcription systems for various bacterial species.
Main Methods:
- In vitro reconstitution of RNA polymerase holoenzymes.
- Heterologous co-overproduction of RNA polymerase subunits in Escherichia coli.
Main Results:
- Both described strategies yield substantial quantities of recombinant RNA polymerase.
- These methods are applicable to any bacterium with known RNA polymerase (rpo) gene sequences.
Conclusions:
- The described methods provide accessible routes for obtaining bacterial RNA polymerase.
- These preparations facilitate the study of species-specific transcription regulation through in vitro systems.
Related Concept Videos
00:43Bacterial RNA Polymerase
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.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
00:43Bacterial RNA Polymerase
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.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
00:58Eukaryotic RNA Polymerases
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.
All three eukaryotic RNAPs require specific transcription factors, of which the...
All three eukaryotic RNAPs require specific transcription factors, of which the...
00:58Eukaryotic RNA Polymerases
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.
All three eukaryotic RNAPs require specific transcription factors, of which the...
All three eukaryotic RNAPs require specific transcription factors, of which the...
01:09Recombinant DNA
Overview
01:47Transcription Initiation
Initiation is the first step of transcription in eukaryotes. Prokaryotic RNA Polymerase (RNAP) can bind to the template DNA and start transcribing. On the other hand, transcription in eukaryotes requires additional proteins, called transcription factors, to first bind to the promoter region in the DNA template. This binding helps recruit the specific RNAP that can assemble on the DNA and start transcription.
The promoters and enhancers and their accessory proteins allow tight regulation of...
The promoters and enhancers and their accessory proteins allow tight regulation of...