NMR studies of translocation of the Zif268 protein between its target DNA Sites.
Zif268 is a zinc-finger protein containing three Cys(2)-His(2)-type zinc-finger domains that bind the target DNA sequence GCGTGGGCG in a cooperative manner. In this work, we characterized translocation of the Zif268 protein between its target DNA sites using NMR spectroscopy. The residual dipolar coupling data and NMR chemical shift data suggested that the structure of the sequence-specific complex between Zif268 and its target DNA in solution is the same as the crystal structure. Using two-dimensional heteronuclear (1)H-(15)N correlation spectra recorded with the fast acquisition method, we analyzed the kinetics of the process in which the Zif268 protein transfers from a target site to another on a different DNA molecule on a minute to hour time scale. By globally fitting the time-course data collected at some different DNA concentrations, we determined the dissociation rate constant for the specific complex and the second-order rate constant for direct transfer of Zif268 from one target site to another. Interestingly, direct transfer of the Zif268 protein between its target sites is >30000-fold slower than corresponding direct transfers of the HoxD9 and the Oct-1 proteins, although the affinities of the three proteins to their target DNA sites are comparable. We also analyzed translocation of the Zif268 protein between two target sites on the same DNA molecules. The populations of the proteins bound to the target sites were found to depend on locations and orientations of the target sites.
Takayama
Yuki
Y
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-1068, USA.
Sahu
Debashish
D
Iwahara
Junji
J
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008279:Magnetic Resonance Imaging; D009682:Magnetic Resonance Spectroscopy; D011506:Proteins / Q000235:genetics; D014178:Translocation, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D011506:Proteins; D004247:DNA