Mutant Escherichia coli arginine repressor proteins that fail to bind l‐arginine, yet retain the ability to bind their normal DNA‐binding sites

Abstract

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

“…We were surprised to find that the apparent affinity of wild-type ArgR for the argF ARG boxes was so low in this experiment; previously, we and others have observed an apparent equilibrium dissociation constant (K d Ј) for ArgR binding to this target of Ϸ1-5 nM (monomer equivalent; Charlier et al, 1992;Tian et al, 1992;Burke et al, 1994). We suspect that this apparently low affinity resulted from a low ArgR-binding-specific activity in the ArgR protein preparation used in this particular experiment.…”

“…This provides a good binding site for ArgR and the mutant proteins ( Fig. 2; Charlier et al, 1992;Tian et al, 1992;Burke et al, 1994). In the presence of L-arginine, Fig.…”

“…1A). Under the same conditions, wild-type ArgR gel-filtered as a hexamer (with an apparent molecular mass of 90-110 kDa in the presence and absence of L-arginine; Burke et al, 1994). ArgRL107K gel-filtered as a trimer (apparent molecular mass 55 kDa Ϯ 8 kDa) in the presence or absence of L-arginine (Fig.…”

“…To test whether expression of ArgRL107A, ArgRNVL107A (containing amino acid substitutions at positions 107, 128 and 129; A, N and V, respectively), ArgRL107K and ArgRNVL107K (with K, N and V at positions 107, 128 and 129, respectively) confer such a phenotype on cells, DS997 and DS998 (ArgR þ and argR derivatives of an Arg þ derivative of DS941, respectively; Burke et al, 1994) were transformed with plasmids expressing each of the ArgR mutants at residue L107. ArgRNVL107A conferred a super-repressor phenotype in both DS997 and DS998; cells required supplementation with L-arginine to allow growth.…”

“…At the high concentrations of protein used in this experiment, the expected relatively poor binding of ArgR and its mutants to a single box in the presence of arginine may be reflected by the instability of the complexes during electrophoresis (and hence the 'smearing forwards' seen in the gel). Elsewhere, it has been shown that ArgR binds to a single ARG box with about 100-fold lower affinity than to two correctly spaced ARG boxes (Charlier et al, 1992;Tian et al, 1992;Burke et al, 1994). No binding of ArgR or its mutants was observed to a single half-site (pSH20), or to two half-sites present in different ARG boxes (pSH25 and pSH26) (data not shown), indicating that the two half-sites of a single ARG box are the minimal requirements for stable binding, though binding can be enhanced by the addition of further half-sites.…”