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Related Concept Videos

Eukaryotic RNA Polymerases00:58

Eukaryotic 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...
Eukaryotic RNA Polymerases00:58

Eukaryotic 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...
Bacterial RNA Polymerase00:43

Bacterial 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...
Bacterial RNA Polymerase00:43

Bacterial 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...
Transcription Initiation01:47

Transcription 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...
Proofreading01:31

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,  it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme

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Related Experiment Video

Updated: Jul 7, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
07:38

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

DNA-independent RNA polymerases.

S Tabor1

  • 1Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary

Poly(A) polymerase adds adenine (AMP) to RNA 3' ends using ATP. This enzyme facilitates RNA labeling for probes and cloning of RNA lacking a poly(A) tail.

Area of Science:

  • Molecular Biology
  • Enzymology

Background:

  • Polyadenylation is a crucial post-transcriptional modification in eukaryotes.
  • Poly(A) polymerase (PAP) is the enzyme responsible for adding the poly(A) tail.
  • Understanding PAP function is vital for RNA manipulation and analysis.

Purpose of the Study:

  • To describe the activity of purified Poly(A) polymerase from E. coli.
  • To detail reaction conditions for polyadenylation.
  • To present applications of PAP in RNA labeling and cloning.

Main Methods:

  • Purification of Poly(A) polymerase from E. coli.
  • In vitro enzymatic assay for AMP incorporation using ATP.
  • RNA 3' end labeling with alpha32ATP.
  • cDNA synthesis using oligo(dT) primer on polyadenylated RNA.

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Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle
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Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Published on: March 5, 2019

Related Experiment Videos

Last Updated: Jul 7, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
07:38

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle
10:05

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Published on: March 5, 2019

Main Results:

  • Poly(A) polymerase efficiently catalyzes the addition of AMP residues to the 3'-hydroxyl terminus of RNA.
  • Optimized reaction conditions were established for PAP activity.
  • Successful labeling of RNA 3' ends was achieved for hybridization probes.
  • Cloning of RNA molecules lacking a poly(A) tail was demonstrated via PAP-mediated tailing and subsequent cDNA synthesis.

Conclusions:

  • Purified E. coli Poly(A) polymerase is a versatile enzyme for RNA modification.
  • PAP is instrumental in generating labeled RNA for molecular hybridization.
  • This method enables the cloning of previously un-clonable RNA lacking poly(A) tails.