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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...
The Eukaryotic Promoter Region02:40

The Eukaryotic Promoter Region

The eukaryotic promoter region is a segment of DNA located upstream of a gene. It contains an RNA polymerase binding site, a transcription start site, and several cis-regulatory sequences.  The proximal promoter region is located in the vicinity of the gene and has cis-regulatory sequences and the core promoter. The core promoter is the binding site for RNA polymerase and is usually located between -35 and +35 nucleotides from the transcription start site. The distal promoter regions are...
Bacterial Transcription01:53

Bacterial Transcription

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:
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...
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...
The Eukaryotic Promoter Region02:40

The Eukaryotic Promoter Region

The eukaryotic promoter region is a segment of DNA located upstream of a gene. It contains an RNA polymerase binding site, a transcription start site, and several cis-regulatory sequences.  The proximal promoter region is located in the vicinity of the gene and has cis-regulatory sequences and the core promoter. The core promoter is the binding site for RNA polymerase and is usually located between -35 and +35 nucleotides from the transcription start site. The distal promoter regions are...

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Related Experiment Video

Updated: May 7, 2026

High-throughput Purification of Affinity-tagged Recombinant Proteins
07:44

High-throughput Purification of Affinity-tagged Recombinant Proteins

Published on: August 26, 2012

T7 RNA polymerase: promoter structure and polymerase binding.

J L Oakley, R E Strothkamp, A H Sarris

    Biochemistry
    |February 6, 1979
    PubMed
    Summary

    T7 RNA polymerase recognizes specific DNA promoter sequences essential for gene transcription. Promoter binding involves DNA melting, influenced by pH and temperature, while enzyme activity requires a free sulfhydryl group.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Bacteriophage T7 RNA polymerase is a crucial enzyme for gene expression in molecular biology.
    • Understanding promoter recognition is key to controlling transcription.
    • The T7 promoter sequences dictate polymerase binding and initiation.

    Purpose of the Study:

    • To present the sequences of two T7 RNA polymerase promoters.
    • To investigate the molecular interactions between T7 RNA polymerase and its promoters.
    • To elucidate the factors affecting polymerase binding and activity.

    Main Methods:

    • DNA sequencing to determine promoter regions.
    • Spectroscopic analysis (hyperchromic blue shift) to study polymerase-promoter interactions.

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  • Enzyme activity assays under varying pH and temperature conditions.
  • Biochemical analysis of enzyme active sites.

    • Main Results:

    • Identical sequences for two T7 promoters from +1 to -15, with a conserved region of dyad symmetry.
    • Promoter A located within gene 1, upstream of the RNase III site.
    • T7 RNA polymerase binding induces DNA base pair melting, evidenced by a blue shift.
    • Binding is inhibited at low pH and temperature; activity is pH-dependent (pKa 7.0).
    • Catalytic activity requires a free sulfhydryl group (pKa ~7.8), distinct from binding.

    Conclusions:

    • The study details specific T7 RNA polymerase promoter sequences and their structural features.
    • T7 RNA polymerase binding involves promoter DNA melting, sensitive to environmental conditions.
    • Enzyme activity and promoter binding are distinct processes with different requirements.