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Related Concept Videos

Cooperative Binding of Transcription Regulators02:13

Cooperative Binding of Transcription Regulators

Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome.  Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form dimers that...
Cooperative Binding of Transcription Regulators02:13

Cooperative Binding of Transcription Regulators

Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome.  Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form dimers that...
Transcription Factors02:16

Transcription Factors

Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
General Transcription Factors01:30

General Transcription Factors

Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

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Related Experiment Video

Updated: Jun 26, 2026

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
06:38

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Published on: February 7, 2019

Quantitative transcription factor binding kinetics at the single-molecule level.

Yufang Wang1, Ling Guo, Ido Golding

  • 1Department of Physics, Princeton University, Princeton, New Jersey, USA.

Biophysical Journal
|January 27, 2009
PubMed
Summary
This summary is machine-generated.

Bacteriophage lambda-repressor CI protein binding to DNA was studied using single-molecule microscopy. Researchers precisely measured association and dissociation rates, finding CI binding to its operator site is independent of flanking DNA length.

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Last Updated: Jun 26, 2026

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
06:38

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Published on: February 7, 2019

Measuring the Kinetics of mRNA Transcription in Single Living Cells
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Measuring the Kinetics of mRNA Transcription in Single Living Cells

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Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates
06:48

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Published on: January 5, 2024

Area of Science:

  • Molecular Biology
  • Biophysics
  • Microscopy

Background:

  • The bacteriophage lambda-repressor CI protein regulates viral gene expression by binding to specific DNA operator sites.
  • Understanding the kinetics of this protein-DNA interaction is crucial for comprehending viral lifecycle control.

Purpose of the Study:

  • To investigate the binding dynamics of bacteriophage lambda-repressor CI protein with its target DNA at the single-molecule level.
  • To determine the association (k(a)) and dissociation (k(d)) rate constants for CI-DNA interactions.
  • To analyze how DNA length, CI dimer concentration, and binding affinity influence these kinetics.

Main Methods:

  • Utilized total internal reflection fluorescence microscopy (TIRFm) to observe single-molecule interactions.
  • Fused the red fluorescent protein (RFP) to the CI protein to monitor binding events.
  • Analyzed thousands of individual association and dissociation events using Poisson and exponential distribution statistics.

Main Results:

  • Observed large, stepwise changes in fluorescence intensity corresponding to CI binding and unbinding.
  • Quantified association and dissociation rate constants (k(a) and k(d)) directly from interval distributions.
  • Demonstrated that CI binding to the operator site is independent of the length of adjacent nonoperator DNA sequences, despite observable non-specific interactions.

Conclusions:

  • Single-molecule TIRFm provides a powerful method for characterizing protein-DNA binding kinetics.
  • The association and dissociation rates of bacteriophage lambda-repressor CI are precisely regulated.
  • CI's specific binding to its operator is robust and not significantly affected by the length of surrounding DNA, highlighting sequence-specific recognition.