Abstract
The trp repressor of Escherichia coli is a dimeric DNA-binding protein that regulates transcription of several operons concerned with tryptophan metabolism. Although heterodimer formation between mutant and wild type subunits occurs readily in vivo, comparable heterodimers could be formed in vitro only under extreme conditions. To explain this difference we analyzed trp repressor dimer formation and dissociation using an in vitro transcription/translation system. Nascent wild type or mutant repressor polypeptides, synthesized in the presence of an excess of a second repressor, were invariably incorporated into heterodimers. In contrast, previously synthesized and assembled wild type dimers appeared to be refractory to dissociation, since they did not form heterodimers. However, previously synthesized mutant dimeric repressors that were defective in tryptophan binding readily dissociated and formed heterodimers. We noted that the ability of a dimeric repressor to dissociate under our conditions correlated inversely with its affinity for tryptophan. Consistent with this conclusion, we found that dissociation of the wild type aporepressor (no added tryptophan) was appreciably more rapid than dissociation of the tryptophan-saturated wild type repressor.
Highlights
$ Supported by National Institutes of Health, National Research Service Award GM11980.Present address: Dept. of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205
The reduced activity of co-expressed wild type and mutant repressors was thought to result from the formation of inactive or partially active heterodimeric repressors composed of one wild type polypeptide and one mutant polypeptide, and theconcomitant reduction in the concentration of wild type homodimers
TM44 repressor was found that heterodimer formationdid occur i n vitro, but was includedin thisexperiment because it has a binding only when repressor mixtures were heat-treated or incubated constant for tryptophan intermediatebetween that of the wild in thepresence of ethanol
Summary
Heterodimer formation between mutant and wild type subunits occurs readily in uivo, comparable heterodimerscould be formed in vitroonly under extreme conditions. To explain this difference we analyzed trp repressor dimer formation and dissociation using an invitro transcription/translation system. Members of one class of dominant negative repressors had amino acid substitutions in the helix-turn-helix domain that presumably reduce operator binding while not affecting dimer formation. Tryptophan presumably stabilized existing repressor dimers that had functional tryptophan binding sites In viewof these observations and the interlocked polypeptide organization revealed by structural studies on the trp repressor, it was not obvious how heterodimer formation could occur so readily in vivo.
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