Mechanism for specificity by HMG-1 in enhanceosome assembly.
Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.
Ellwood
K B
KB
Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA.
Yen
Y M
YM
Johnson
R C
RC
Carey
M
M
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins / Q000235:genetics*; D009154:Mutation; D009690:Nucleic Acid Conformation; D009698:Nucleoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics
D002352:Carrier Proteins; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D009698:Nucleoproteins; D011994:Recombinant Proteins; D004247:DNA