The first high-mobility-group box of upstream binding factor assembles across-over DNA junction by basic residues

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Upstream binding factor (UBF) is a eukaryotic RNA polymerase I-specific transcription factor.
• The ubfHMG box 1 motif is critical for UBF's DNA-binding and assembly capabilities.
• This motif can bind multiple DNA duplexes, forming crossover DNA junctions.

Purpose of the Study:

• To investigate the molecular basis of the ubfHMG box 1 motif's DNA-assembling activity.
• To determine which residues and domains are essential for forming DNA crossover junctions.
• To elucidate the mechanism of UBF-mediated DNA complex formation.

Main Methods:

• Extensive mutagenesis analysis of the ubfHMG box 1 motif.
• Mobility shift assays to assess DNA-binding and assembly.
• Site-directed mutagenesis of conserved hydrophobic and basic residues.
• Analysis of dimerization domain mutations.

Main Results:

• Mutations in hydrophobic and aromatic residues did not significantly impair DNA-assembling activity.
• Alterations in basic residues within helices 1 and 2, and the N-terminal strand, severely affected DNA-assembling activity.
• Non-specific DNA binding persisted even after mutations affecting DNA assembly.
• Mutation of a hydrophobic residue in the dimerization domain inhibited peptide association but not DNA-assembling activity.

Conclusions:

• The DNA-assembling activity of ubfHMG box 1 relies on interactions with the DNA backbone, mediated by basic residues.
• Conserved hydrophobic and aromatic residues are not directly involved in DNA-protein interactions for junction formation.
• The UBF-DNA complex is not formed through the association of individual DNA-bound peptides.