Nucleic Acids Research, 1993, Vol. 21, No. 8 1727-1734
© 1993
MOLECULAR BIOLOGY |
Role of cysteine62 in DNA recognition by the P50 subunit of NF-xB
School of Biological and Medical Sciences, Irvine Building, University of St Andrews St Andrews, Fife KY16 9AL, UK 1Glaxo Institute for Molecular Biology 14, chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland
* To whom correspondence should be addressed
Received February 19, 1993. Revised March 19, 1993. Accepted March 19, 1993.
A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunlt of NF-xB. Differential labelling with [14C] iodoacetate has Identified a conserved cystelne residue, Cys62, that was protected from modification by the presence of an ollgonucleotide containing the specific recognition site of the protein. To determine the Importance of this cystelne residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis Indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an Intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotlde variants of the DNA recognition site and nonspecific E.coil DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was Impaired in Its ability to discriminate between specific and nonspecific DNA. Thus the sulphydryl group of Cys62 Is an important determinant of DNA recognition by the p50 subunit of NF-xB.
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