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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.
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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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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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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Transfer RNA Synthesis02:36

Transfer RNA Synthesis

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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
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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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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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A Sirtuin-Dependent T7 RNA Polymerase Variant.

Yongan Wang1, Yanli Ji2, Lin Sun1

  • 1Frontiers Science Center for Synthetic Biology, Tianjin Key Laboratory of Function and Application of Biological Macromolecular Structures, School of Life Sciences, Tianjin University, Tianjin 300072, China.

ACS Synthetic Biology
|December 20, 2023
PubMed
Summary
This summary is machine-generated.

Scientists engineered a T7 RNA polymerase (T7RNAP) variant using genetic code expansion. This modified T7RNAP requires sirtuin activity to function, linking gene transcription to sirtuin expression and NAD levels.

Keywords:
T7 RNA polymerasedeacetylationgenetic code expansionlysine acetylationsirtuintranscription

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biotechnology

Background:

  • Transcriptional regulation is crucial for cellular homeostasis.
  • Controlling gene expression is a key goal in synthetic biology and bioengineering.
  • Novel methods for transcriptional control are actively sought.

Purpose of the Study:

  • To develop a novel transcriptional control system.
  • To engineer a T7 RNA polymerase (T7RNAP) variant responsive to cellular regulatory mechanisms.
  • To link gene transcription to sirtuin activity and NAD+ availability.

Main Methods:

  • Genetic code expansion was employed to create a T7RNAP variant.
  • An essential lysine (K631) in the T7RNAP catalytic core was replaced with Nε-acetyl-l-lysine (AcK).
  • The functionality of the T7RNAP variant was tested in bacterial, mammalian, and in vitro systems.

Main Results:

  • The engineered T7RNAP variant exhibited restored enzymatic activity dependent on deacetylase activity of NAD-dependent sirtuins.
  • Sirtuin-dependent transcription of target genes was achieved in live cells (bacteria and mammalian) and in vitro.
  • The T7RNAP variant successfully linked gene transcription to sirtuin expression and NAD availability.

Conclusions:

  • A novel, sirtuin-dependent T7RNAP variant was successfully engineered.
  • This variant enables a new mode of transcriptional regulation controllable by sirtuin activity.
  • The engineered T7RNAP holds potential for applications in synthetic biology, bioengineering, and studying cellular metabolism.