The salt dependence of DNA recognition by NF-kappaB p50: a detailed kinetic analysis of the effects on affinityand specificity.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Hart DJ,Speight RE,Cooper MA,Sutherland JD,Blackburn JM

doi

10.1093/nar/27.4.1063

subject

Has Abstract

pub_date

1999-02-15 00:00:00

pages

1063-9

issue

4

eissn

0305-1048

issn

1362-4962

pii

gkc229

journal_volume

27

pub_type

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