Specific protein-DNA and protein-protein interaction in the hig gene system, a plasmid-borne proteic killer gene system of plasmid Rts1

The hig (host inhibition of growth) gene system of plasmid Rts1 belongs to the plasmid-encoded proteic killer gene family. Among the proteic killer ge nes described so far. hig is unique in that the toxin gene (higB) exists up stream of the antidote gene (higA). There are two promoters in the hig locu s. Phig and PhigA. and only the former, which expresses both higB and higA genes, is negatively controlled by HigA and HigB proteins. In this study. w e purified HigA protein bq means of GST fusion. The electrophoretic mobilit y shift assay using the purified protein revealed that HigA specifically bo und to the Phig region, but not to PhigA. The HigA-binding sequence was det ermined by DNase I footprinting assay to be a 56-bp sequence that completel y covered the -35 and -10 boxes of Phig. The presence of two inverted repea ts in the binding sequence and the identification of a dimer form of HigA b y cross-linking experiment suggested that the protein bound to the Phig reg ion as a dimer. HigB was purified as a GST fusion protein as well, though i t was achieved only in the: presence of HigA. HigA and GST-HigB formed a hi ghly stable complex where the two proteins were present in an equimolar rat io. (C) 2001 Academic Press.