DNA-bending properties of TF1.
Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of DNA-binding proteins that includes Escherichia coli HU and integration host factor, IHF. A gel electrophoretic retardation method has been used to show that a TF1 dimer binding to one of its preferred sites in (5-hydroxymethyl)uracil (hmUra)-containing DNA sharply bends the latter. In fact, the DNA-bending properties of TF1 and E. coli IHF are indistinguishable. Substitutions at amino acid 61 in the DNA-binding "arm" of TF1 are known to affect DNA-binding affinity and site selectivity. Experiments described here show that these substitutions also affect DNA bending. The selectivity of TF1 binding is very greatly diminished and the affinity is reduced when hmUra is replaced in DNA by thymine (T). An extension of the gel retardation method that permits an analysis of DNA bending by non-specifically bound TF1 is proposed. Under the assumptions of this analysis, the reduced affinity of TF1 for T-containing DNA is shown to be associated with bending that is still sharp. The analysis of the TF1-DNA interaction has also been extended by hydroxyl radical (.OH) and methylation interference footprinting at two DNA sites. At each of these sites, and on each strand, TF1 strongly protects three segments of DNA from attack by OH. Patches of protected DNA are centered approximately ten base-pairs apart and fall on one side of the B-helix. Methylation in either the major or minor groove in the central ten base-pairs of the two TF1 binding sites quantitatively diminishes, but does not abolish, TF1 binding. We propose that multiple protein contacts allow DNA to wrap around the relatively small TF1 dimer, considerably deforming the DNA B-helix in the process.
Schneider
G J
GJ
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Sayre
M H
MH
Geiduschek
E P
EP
D001412:Bacillus subtilis; D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006878:Hydroxides; D017665:Hydroxyl Radical; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015183:Restriction Mapping; D014764:Viral Proteins
D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D006878:Hydroxides; C059361:TF1 DNA-binding protein, Bacteriophage SPO1; D014764:Viral Proteins; D017665:Hydroxyl Radical; D003850:Deoxyribonuclease I