HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.
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 35S-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 (Kd = 28 nM) 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.
Webster
C I
CI
Cambridge Centre for Molecular Recognition and Department of Plant Sciences, University of Cambridge, UK.
Packman
L C
LC
Gray
J C
JC
D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004270:DNA, Circular / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D004926:Escherichia coli / Q000378:metabolism; D025741:HMGA1a Protein; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D018532:Pisum sativum / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D010970:Plastocyanin / Q000235:genetics*; D011485:Protein Binding
D002352:Carrier Proteins; D004270:DNA, Circular; C107092:HMGA1a Protein, plant; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D010940:Plant Proteins; D025741:HMGA1a Protein; D010970:Plastocyanin