| Autor: |
Gursky, G.V., Surovaya, A.N., Kurochkin, A.V., Chernov, B.K., Volkov, S.K., Kirpichnikov, M.P. |
| Zdroj: |
Journal of Biomolecular Structure and Dynamics; August 1992, Vol. 10 Issue: 1 p15-33, 19p |
| Abstrakt: |
AbstractIn the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage ero repressor to a 17 base pair operator 0R3. We have found that three minor groove-binding antibiotics, dis-tamycin A netropsin and sibiromycin, compete effectively with the ero for binding to the operator 0R3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace ero repressor from the operator 0R3. The binding of crorepressor to the 0R3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270–290 nm. The CD spectral profile of the cro-0R3 mixture in the presence of distamycin Acan be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270–360 nm aproaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of crorepressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of crorepressor to the synthetic operator from competition-type experiments. The binding constant of ero repressor to the Or3 is found tobe (6± 1) · 106M−1at 20 °C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F. |
| Databáze: |
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