Open AccessHMG‐1 enhances HMG‐I/Y binding to an A/T‐rich enhancer element from the pea plastocyanin gene
Author(s) -
Webster Carl I.,
Packman Leonard C.,
Gray John C.
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2001.02191.x
Subject(s) - plastocyanin , enhancer , high mobility group , microbiology and biotechnology , biology , dna , hmg coa reductase , dna binding protein , binding site , chemistry , gene , biochemistry , gene expression , transcription factor , enzyme , photosystem i , chloroplast , reductase
High‐mobility‐group proteins HMG‐1 and HMG‐I/Y bind at overlapping sites within the A/T‐rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG‐1 enhanced the binding of HMG‐I/Y to a 31‐bp region (P31) of the enhancer. Circularization assays showed that HMG‐1, but not HMG‐I/Y, was able to bend a linear 100‐bp DNA containing P31 so that the ends could be ligated. HMG‐1, but not HMG‐I/Y, showed preferential binding to the circular 100‐bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG‐1 was not responsible for enhanced binding of HMG‐I/Y. Direct interaction of HMG‐I/Y and HMG‐1 in the absence of DNA was demonstrated by binding of 35 S‐labeled proteins to immobilized histidine‐tagged proteins, and this was due to an interaction of the N‐terminal HMG‐box‐containing region of HMG‐1 and the C‐terminal AT‐hook region of HMG‐I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG‐1 had an affinity for immobilized HMG‐I/Y ( K d = 28 n m ) similar to that for immobilized P31 DNA. HMG‐1‐enhanced binding of HMG‐I/Y to the enhancer element appears to be mediated by the formation of an HMG‐1–HMG‐I/Y complex, which binds to DNA with the rapid loss of HMG‐1.