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Contacts between the factor TUF and RPG sequences.

M L Vignais1, J Huet, J M Buhler

  • 1Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.

The Journal of Biological Chemistry
|August 25, 1990
PubMed
Summary
This summary is machine-generated.

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The yeast TUF factor specifically binds RPG sequences, crucial for gene regulation. Researchers detailed TUF

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The yeast TUF factor is a transcriptional regulator that binds to RPG-like sequences.
  • These sequences are involved in diverse cellular functions, including enhancer, silencer, and telomere regulation.

Purpose of the Study:

  • To characterize the molecular interactions between the yeast TUF factor and its optimal binding sequence, rpg-1.
  • To determine the precise DNA-binding interface and the affinity of TUF for its target sequence.

Main Methods:

  • Gel DNA-binding assays were employed to study TUF-DNA interactions.
  • Methylation protection and mutagenesis experiments were used to identify critical base pairs within the RPG sequence.
  • Quantitative band shift analysis determined the equilibrium dissociation constant (Kd) for the TUF-rpg-1 complex.

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Main Results:

  • TUF binding engages up to 10 base pairs of the rpg-1 sequence, with 8 identified as important for binding.
  • Mutagenesis and methylation protection pinpointed critical contact points, including a central GC pair at position 5.
  • The consensus binding sequence was determined as RCAAYCCRYNCAYY.
  • The equilibrium dissociation constant (Kd) for TUF and rpg-1 was found to be 1.3 x 10(-11) M, indicating high binding affinity.

Conclusions:

  • The yeast TUF factor exhibits high specificity and affinity for its RPG target sequences.
  • Detailed characterization provides insights into the molecular basis of TUF-mediated gene regulation at enhancers, silencers, and telomeres.