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Related Concept Videos

Eukaryotic RNA Polymerases00:58

Eukaryotic RNA Polymerases

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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.
All three eukaryotic RNAPs require specific transcription factors, of which the...
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Transcription Initiation01:47

Transcription Initiation

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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...
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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.
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...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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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.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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Bacterial Transcription01:53

Bacterial Transcription

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RNA polymerase (RNAP) carries out DNA-dependent RNA synthesis in both bacteria and eukaryotes. Bacteria do not have a membrane-bound nucleus. So, transcription and translation occur simultaneously, on the same DNA template.
Transcription can be divided into three main stages, each involving distinct DNA sequences to guide the polymerase. These are:
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RNA Polymerase II Accessory Proteins02:36

RNA Polymerase II Accessory Proteins

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Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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Engineered Stop and Go T7 RNA Polymerases.

Zachary T Baumer1, Matilda S Newton1, Lina Löfstrand1

  • 1Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, Colorado 80305, United States.

ACS Synthetic Biology
|November 29, 2024
PubMed
Summary
This summary is machine-generated.

Scientists developed ligand-activated RNA polymerases (LARPs) that are precisely controlled by indoles. This breakthrough enables "stop and go" gene expression for synthetic biology applications.

Keywords:
RNA polymeraseallosterydynamic metabolic controlintercellular signalingprotein engineering and designsynthetic biology

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Area of Science:

  • Synthetic Biology
  • Enzyme Engineering
  • Molecular Biology

Background:

  • Precise enzyme activation is crucial for synthetic biology.
  • Unregulated T7 RNA polymerase can halt bacterial growth.
  • Natural enzymes are regulated by endogenous metabolites.

Purpose of the Study:

  • To engineer T7 RNA polymerases (T7 RNAP) activated by physiological metabolites.
  • To create a controllable gene expression system using indoles.
  • To develop a "stop and go" platform for synthetic biology.

Main Methods:

  • Rational design and directed evolution of T7 RNAP variants.
  • Characterization of indole-activated enzyme kinetics (EC50).
  • Demonstration of indole-mediated gene expression control in various contexts.

Main Results:

  • Identified T7 RNAP variants with minimal basal activity and 29-fold induction by indole (EC50 = 344 µM).
  • Indoles regulated T7-dependent gene expression exogenously, endogenously, and intercellularly.
  • Demonstrated indole-dependent bacteriophage propagation and engineered ligand-activated RNA polymerases (LARPs).

Conclusions:

  • Ligand-activated RNA polymerases (LARPs) offer a novel, chemically inducible platform.
  • LARPs provide precise "stop and go" control for synthetic biology.
  • This system is portable across bacterial species and applicable to synthetic cocultures.