The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity
Hart, D., Speight, R.E., Cooper, M.A., Sutherland, J.D., & Blackburn, J.M. (1999) The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity. Nucleic Acids Research, 27(4), pp. 1063-1069.
The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.
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|Item Type:||Journal Article|
|Additional Information:||Articles free to read on journal website|
|Divisions:||Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > QUT Faculties and Divisions > Science & Engineering Faculty
|Deposited On:||22 Apr 2014 04:51|
|Last Modified:||29 Jan 2015 01:42|
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