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Related Experiment Videos

Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

N Dean1, A J Berk

  • 1Molecular Biology Institute, UCLA, Los Angeles, CA 90024.

Nucleic Acids Research
|December 10, 1987
PubMed
Summary

Mammalian transcription factor IIIC (TFIIIC) separates into two functional components, TFIIIC1 and TFIIIC2, essential for tRNA and VA RNA gene transcription. TFIIIC2 specifically binds the B block promoter region, crucial for pre-initiation complex formation.

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biochemistry

Background:

  • Mammalian transcription factor IIIC (TFIIIC) is vital for transcribing tRNA and adenovirus VA RNA genes.
  • Previous studies indicated TFIIIC activity could be separated into two functional components.

Purpose of the Study:

  • To confirm and further characterize the two functional components of mammalian TFIIIC.
  • To elucidate the specific roles and DNA-binding properties of these TFIIIC components.

Main Methods:

  • Anion exchange FPLC chromatography to separate TFIIIC components.
  • Sequence-specific DNA affinity chromatography to isolate TFIIIC fractions.
  • DNase I protection assays to determine DNA-binding sites.
  • Template competition and rescue assays to assess pre-initiation complex formation.

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Main Results:

  • TFIIIC activity was successfully separated into two components, TFIIIC1 and TFIIIC2, using both FPLC and DNA affinity chromatography.
  • TFIIIC2 was identified as the DNA-binding component, specifically recognizing and binding to the 3' segment (B block) of the internal promoter.
  • TFIIIC2 was shown to be the limiting and titratable factor required for stable pre-initiation complex formation on the VAI RNA gene.

Conclusions:

  • Mammalian transcription requires at least three proteins, including TFIIIC1 and TFIIIC2, in addition to RNA polymerase III for VA RNA and tRNA gene transcription.
  • TFIIIC2 plays a critical role in promoter recognition and initiation complex assembly through specific binding to the B block promoter element.