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Related Experiment Videos

Intramolecular triplex potential sequence within a gene down regulates its expression in vivo.

P S Sarkar1, S K Brahmachari

  • 1Molecular Biophysics Unit, Indian Institute of Science, Bangalore.

Nucleic Acids Research
|November 11, 1992
PubMed
Summary

Intramolecular triple helix sequences in genes can inhibit gene expression. This study shows these structures block RNA polymerase, reducing protein production.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Polypurine/polypyrimidine sequences can form intramolecular triple helix structures under specific conditions (e.g., low pH, torsional stress).
  • These sequences are found in gene regulatory and coding regions, with potential roles in transcriptional control.
  • The in vivo function of triple helix structures within coding regions remains largely unexplored.

Purpose of the Study:

  • To investigate whether sequences forming intramolecular triple helices within a gene's coding region can be transcribed by Escherichia coli RNA polymerase.
  • To determine the effect of such triple helix-forming sequences on gene expression in vivo.
  • To explore the potential role of DNA secondary structures in regulating gene transcription elongation.

Main Methods:

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  • Engineering a plasmid (pSBT1) containing a 38 bp polypurine/polypyrimidine sequence motif designed to form an intramolecular triple helix within the beta-galactosidase gene coding region.
  • Utilizing codon degeneracy to create a control plasmid (pSBmT12) with an identical amino acid sequence but lacking the triple helix motif.
  • Transforming E. coli JM109 cells with both constructs and comparing beta-galactosidase expression levels and transcript integrity.

Main Results:

  • A construct (pSBT1) with the triple helix potential sequence showed an 80% inhibition of beta-galactosidase expression compared to the control (pSBmT12).
  • Truncated beta-galactosidase transcripts were detected in cells expressing pSBT1, but not in cells expressing pSBmT12.
  • The presence of the polypurine/polypyrimidine sequence motif capable of forming a triple helix significantly impacted gene expression.

Conclusions:

  • A putative triple helix-forming sequence within a gene's coding region can down-regulate gene expression.
  • Triple helix formation appears to partially block transcription elongation in vivo.
  • These findings suggest a novel mechanism for gene regulation involving DNA secondary structures within coding sequences.