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Related Experiment Videos

Long-range interactions at the HO promoter

H J McBride1, R M Brazas, Y Yu

  • 1Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah Health Sciences Center, Salt Lake City 84132, USA.

Molecular and Cellular Biology
|May 1, 1997
PubMed
Summary

The SWI5 gene and Pho2p protein cooperatively bind to two sites in the yeast HO promoter. Both sites are essential for HO gene activation, with Swi5p mediating long-range interactions.

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Area of Science:

  • Molecular Biology
  • Yeast Genetics
  • Gene Regulation

Background:

  • The SWI5 gene encodes a zinc finger DNA-binding protein crucial for yeast HO gene transcription.
  • Swi5p (SWI5 protein) and Pho2p (a homeodomain protein) are known to interact and bind to the HO promoter.
  • Previous studies indicated that Swi5p binding sites A and B have distinct roles and dependencies on Pho2p.

Purpose of the Study:

  • To investigate the cooperative binding of Swi5p and Pho2p to both identified sites (A and B) within the yeast HO promoter.
  • To elucidate the functional requirement of these Swi5p binding sites for HO gene expression, particularly in the presence and absence of Pho2p.
  • To propose and provide evidence for a model explaining the mechanism of HO gene activation involving Swi5p interactions.

Main Methods:

Related Experiment Videos

  • Site-directed mutagenesis was used to create point mutations in the Swi5p binding sites (A and B) of the HO promoter.
  • Analysis of HO gene expression levels in wild-type and mutant yeast strains, including pho2 mutants.
  • Deletion analysis of the SWI5 gene to identify functional domains involved in PHO2-independent activation.

Main Results:

  • Swi5p and Pho2p cooperatively bind to both HO promoter sites A and B, with differential binding characteristics.
  • Stronger mutations in Swi5p binding sites rendered the HO promoter inactive, highlighting the absolute requirement for both sites.
  • The HO promoter remained inactive in pho2 mutants even with intact Swi5p binding sites, underscoring Pho2p's role in certain conditions.
  • A specific region near the N-terminus of Swi5p was identified as essential for PHO2-independent HO activation.

Conclusions:

  • Both Swi5p binding sites in the HO promoter are indispensable for HO gene expression.
  • A model involving physical interaction between Swi5p proteins bound to distant sites (A and B) is proposed to explain HO activation.
  • The N-terminal region of Swi5p plays a critical role in mediating these long-range interactions for PHO2-independent activation.