THE HMG DOMAIN OF THE ROX1 PROTEIN MEDIATES REPRESSION OF HEM13 THROUGH OVERLAPPING DNA-BINDING AND OLIGOMERIZATION FUNCTIONS

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required f or the repression of genes expressed under anaerobic conditions, ROX1 belongs to a family of DNA binding proteins which contain the high mob ility group motif (HMG domain), To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coil as fu sions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA, Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domai n were able to bind to specific target DNA sequences, In contrast, ROX 1 proteins which contained deletions within the HMG domain were no lon ger capable of binding to DNA, The oligomerization of ROX1 in vitro wa s demonstrated using affinity-purified GST-ROX1 protein and ROX1 label led with [S-35]methionine. Using various ROX1 protein derivatives we w ere able to demonstrate that the domain required for ROX1-ROX1 interac tion resides within the N-terminal 100 amino acids which constitute th e HMG domain, Therefore, the HMG domain is required for both DNA bindi ng activity and oligomerization of ROX1.