A bipartite DNA-binding domain in yeast Reb1p

Area of Science:

• Molecular Biology
• Yeast Genetics
• Protein Structure-Function Relationships

Background:

• The REB1 gene is crucial for Saccharomyces cerevisiae growth.
• Reb1p, the protein it encodes, binds DNA in transcriptional control regions.
• Understanding Reb1p's DNA-binding domain is key to its function.

Purpose of the Study:

• To define the DNA-binding domain of the yeast REB1 protein (Reb1p).
• To investigate the structural features of Reb1p essential for DNA binding.
• To compare REB1 homologs in different yeast species.

Main Methods:

• Constructing 5' and 3' deletions in the REB1 coding region for bacterial expression.
• Cloning the REB1 homolog from Kluyveromyces lactis via genetic complementation.
• Performing DNA footprinting assays with truncated and homologous Reb1p proteins.

Main Results:

• Nearly 400 amino acids at the C-terminus of Reb1p are required for maximal DNA binding.
• The Kluyveromyces lactis REB1 gene complements a Saccharomyces cerevisiae REB1 deletion.
• Two conserved, myb-like regions within Reb1p, separated by variable amino acids, form the DNA-binding domain.

Conclusions:

• The DNA-binding domain of Reb1p is composed of two distinct myb-like regions.
• These regions can accommodate significant variation in the intervening sequence, suggesting a flexible structure.
• Reb1p likely folds to bring these two binding components into proximity to interact with DNA.