Subunit exchange and the role of dimer flexibility in DNA binding by the Fis protein.
Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.
Merickel
Stacy K
SK
Department of Biological Chemistry, School of Medicine, Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California 90095, USA.
Sanders
Erin R
ER
Vázquez-Ibar
José Luis
JL
Johnson
Reid C
RC
D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D035021:Factor For Inversion Stimulation Protein; D005452:Fluoresceins / Q000737:chemistry; D034982:Integration Host Factors; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002352:Carrier Proteins; D035021:Factor For Inversion Stimulation Protein; D005452:Fluoresceins; D034982:Integration Host Factors; D021122:Protein Subunits; D012996:Solutions; C037041:fluorescein 5-maleimide; D004247:DNA; D003545:Cysteine