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Related Concept Videos

Cooperative Binding of Transcription Regulators02:13

Cooperative Binding of Transcription Regulators

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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...
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Cooperative Binding of Transcription Regulators02:13

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Transcription Factors02:16

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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...
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Transcription Elongation Factors02:35

Transcription Elongation Factors

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Transcription elongation is a dynamic process that alters depending upon the sequence heterogeneity of the DNA being transcribed. Hence, it is not surprising that the elongation complex's composition also varies along the way while transcribing a gene.
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA...
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Cooperative Allosteric Transitions01:58

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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
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Related Experiment Video

Updated: Jan 30, 2026

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach
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NextPBM: a platform to study cell-specific transcription factor binding and cooperativity.

Nima Mohaghegh1, David Bray1,2, Jessica Keenan1,2

  • 1Department of Biology and Biological Design Center, Boston University, Boston, MA, USA.

Nucleic Acids Research
|January 19, 2019
PubMed
Summary

We developed nextPBM, a high-throughput method using native proteins to study transcription factor (TF) DNA binding, accounting for cell-specific modifications and cofactors. This approach reveals how TF binding is influenced by phosphorylation and cooperativity.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • High-throughput (HT) in vitro methods are crucial for studying transcription factor (TF) DNA binding and gene regulation.
  • Current methods lack the ability to incorporate endogenous proteins, thus failing to account for cell-specific post-translational modifications (PTMs) and cooperative cofactors.

Purpose of the Study:

  • To introduce the high-throughput nextPBM (nuclear extract protein-binding microarray) approach for analyzing native TF DNA binding.
  • To investigate the impact of PTMs and cell-specific cofactors on TF binding using endogenous proteins.

Main Methods:

  • Integration of immune-depletion and phosphatase treatment into the nextPBM pipeline.
  • Analysis of PU.1/SPI1 and IRF8 binding in human monocytes.
  • Delineation of DNA-sequence determinants for TF cooperativity.

Main Results:

  • Characterization of TF binding, considering PTMs and cofactors.
  • Identification of DNA-sequence elements governing cooperativity between PU.1 and IRF8.
  • Correlation of PU.1 binding affinity with enhancer status and cofactor presence.

Conclusions:

  • nextPBM enables the study of native TF DNA binding, incorporating crucial regulatory factors.
  • The method can discover cell-specific cofactors and characterize TF cooperativity.
  • nextPBM facilitates the design of synthetic cooperative DNA elements.