The first high-mobility-group box of upstream binding factor assembles across-over DNA junction by basic residues.
Upstream binding factor (UBF) is a eukaryotic RNA polymerase I-specific transcription factor. Its predominant DNA-binding motif, ubfHMG box 1, preserves DNA assembling activity that can bind two or more DNA duplexes simultaneously to form a crossover DNA junction. Here we investigate the basis of crossover DNA-assembling activity of ubfHMG box 1 by extensive mutagenesis analyses and mobility shift assay. Although the ubfHMG box 1 preserves a high mobility group (HMG) core structure, changing a number of the consensus hydrophobic and aromatic residues to alanine did not inhibit its crossover-assembling activity. This indicates that these residues do not directly participate in protein-DNA interaction. However, altering a series of basic residues in the helices 1 and 2 regions or the N-terminal extended strand of the ubfHMG box 1 motif had severe effects on DNA-assembling activity; however, certain non-specific DNA binding activity still remained. This suggests that the ubfHMG box 1 motif might extensively contact the backbone of a crossover junction through its multiple basic residues. Mutating a hydrophobic residue in the terminal dimerization domain inhibited the association of truncated Xenopus UBF, but had little effect on its crossover-assembling activity. This indicates that the UBF-crossover DNA complex is not established by the association of individual DNA-bound peptides.
Hu
C H
CH
Institute of Marine Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 20224, Taiwan, Republic of China. chhu@ntou66.ntou.edu.tw
Wang
J M
JM
Tseng
H B
HB
D000595:Amino Acid Sequence; D003434:Crossing Over, Genetic; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins
D006609:High Mobility Group Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C062332:UBFT protein, Xenopus; D029867:Xenopus Proteins; D004247:DNA