Binding stoichiometry (original) (raw)

2011

Abstract

<b>Copyright information:</b>Taken from "TAR-RNA recognition by a novel cyclic aminoglycoside analogue"Nucleic Acids Research 2006;34(12):3599-3608.Published online 19 Jul 2006PMCID:PMC1524922.© 2006 The Author(s) The function ( − )/( − ) is plotted versus the concentration of the aminoglycoside analogue in each lane of the PACE gel. is the retardation distance in each lane, is the retardation distance of the TAR-RNA without and is the retardation distance of the TAR-RNA completely bound to (concentration of = 500 µM). The function ( − )/( − ) can be expressed in terms of molar fractions of bound and free RNA, according to the procedure described by Cilley and Williamson (). Different binding models were assumed and the theoretical dependence of ( − )/( − ) on the concentration of was calculated in each case from the molar fractions assuming constant concentration of in each lane. The experimental data (red dots) were fitted to the theoretical functions using IgorPro to derive the binding stoichiometry for the TAR/ complex. Six different models were considered: (i) one molecule of the TAR-RNA binds one molecule of (yellow line); (ii) one molecule of the TAR-RNA binds two molecules of with the same (green line); (iii) two molecules of the RNA bind one molecule of (magenta); (iv) one molecule of the TAR-RNA binds two molecules of with different (blue line); (v) one molecule of the TAR-RNA binds one molecule of (); subsequently the TAR/ complex dimerizes () (close to the yellow line, not shown); (vi) two molecules of the RNA bind one molecule of (2 ); subsequently two molecules of are recruited by the RNA dimer for a total of two RNA molecules and three molecules of () (red line; ≈ 20 µM; ≈ 3 mM). The best fit is obtained for case (f), which supports the stoichiometry inferred from the NMR data.

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