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Related Experiment Videos

Activating transcription from single stranded DNA

T Tomonaga1, D Levens

  • 1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Proceedings of the National Academy of Sciences of the United States of America
|June 11, 1996
PubMed
Summary
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Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds single-stranded DNA, influencing gene transcription in vivo. This study demonstrates hnRNP K

Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • Sequence-specific regulators of eukaryotic gene expression typically target double-stranded DNA.
  • Proteins binding single-stranded DNA have lacked in vivo evidence for transcriptional roles.
  • Heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds the single-stranded CT-element of the human c-myc gene in vitro.

Purpose of the Study:

  • To determine if sequence-specific single-strand DNA binding proteins can locate cognate elements and modulate transcription in vivo.
  • To investigate the in vivo DNA binding and transcriptional regulatory role of hnRNP K.

Main Methods:

  • Studied hnRNP K's interaction with the single-stranded CT-element of the human c-myc gene.
  • Fused hnRNP K with the VP16 transactivation domain to monitor in vivo DNA binding via reporter gene activation.

Related Experiment Videos

  • Engineered CT-elements to overlap with lexA operators to assess competition for binding.

    • Main Results:

    • hnRNP K-VP16 fusion protein transactivated circular but not linear CT-element reporters, indicating recognition of single-stranded DNA formed by supercoiling.
    • Addition of lexA protein abrogated hnRNP K binding, suggesting it prevents single-strand formation.
    • Demonstrated that hnRNP K functions as a single-strand DNA binding protein in vivo.

    Conclusions:

    • hnRNP K is a sequence-specific single-strand DNA binding protein that functions in vivo.
    • DNA segments can influence adjacent gene transcription through duplex-single strand interconversions.
    • This provides a mechanism for single-strand DNA binding proteins to regulate gene expression.