Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine

Exogenous leucine affects the expression of a number of different operons in Escherichia coli. For at least some of these operons, the leucine-related effect is mediated by a protein called Lrp (Leucine-responsive regulatory protein). The purification of Lrp to near homogeneity is described. Lrp is a moderately abundant, basic protein composed of two subunits of molecular mass 18.8 kDa each. In addition, the corresponding protein was purified from a strain having a mutation within the gene that encodes Lrp (lrp). This mutation (lrp-1) causes high constitutive expression of ilvIH, one of the operons controlled by Lrp (Platko, J. V., Willins, D.A., and Calvo, J.M. (1990) J. Bacteriol. 172, 4563-4570). The Lrp-1 and Lrp proteins have similar physical properties, but they show some differences in the characteristics with which they bind DNA upstream of the ilvIH promoter. The nucleotide sequences of the lrp and lrp-1 genes differ by only a single nucleotide, a C to G change that would substitute a Glu for an Asp at amino acid 114. Lrp has some amino acid sequence similarity to AsnC, a protein that regulates asnA expression (Kolling, R., and Lother, H. (1985) J. Bacteriol. 164, 310-315).     

Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli

Lrp is a global regulatory protein in Escherichia coli that activates expression of more than a dozen operons and represses expression of another dozen. For some operons, exogenous leucine reduces the extent of Lrp action, for others it potentiates the effect of Lrp, and for yet other operons it has no effect. In an effort to understand how leucine affects Lrp-mediated expression, we examined Lrp self-association and the effect of leucine on self-association using light scattering, chemical cross-linking, and analytical ultracentrifugation. The following results were obtained. (i) Lrp self-associates to a hexadecamer and octamer with the predominant species being hexadecamer at microM concentrations. (ii) Lrp undergoes a leucine-induced dissociation of hexadecamer to octamer. (iii) A mutant Lrp lacking 11 amino acid residues at the C terminus does not form higher-order oligomers, suggesting that the C terminus is involved in subunit association. (iv) At nM concentrations, Lrp dissociates to a dimer. It is proposed that leucine regulates the equilibrium between Lrp oligomers and thus Lrp occupancy of sites within different operons, leading to diverse regulatory patterns.     

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.     

The ilvIH operon of Escherichia coli is positively regulated.

The ilvIH operon of Escherichia coli (located near min 2) encodes acetohydroxyacid synthase III, an isozyme involved in branched-chain amino acid biosynthesis. A strain with lacZ fused to the ilvIH promoter was constructed. Transposon Tn10 was introduced into this strain, and tetracycline-resistant derivatives were screened for those in which ilvIH promoter expression was markedly reduced. In one such derivative, strain CV1008, beta-galactosidase expression was reduced more than 30-fold. The transposon giving rise to this phenotype inserted near min 20 on the E. coli chromosome. Extract from a wild-type strain contains a protein, the IHB protein, that binds to two sites upstream of the ilvIH promoter (E. Ricca, D. A. Aker, and J. M. Calvo, J. Bacteriol. 171:1658-1664, 1989). Extract from strain CV1008 lacks IHB-binding activity. These results indicate that the IHB protein is a positive regulator of ilvIH operon expression. The gene that encodes the IHB protein, ihb, was cloned by complementing the transposon-induced mutation. Definitive evidence that the cloned DNA encodes the IHB protein was provided by determining the sequence of more than 17 amino acids at the N terminus of the IHB protein and comparing it with the nucleotide sequence. A mutation that prevents repression of the ilvIH operon by leucine in vivo and that alters the DNA-binding characteristics of the IHB protein in vitro was shown to be an allele of the ihb gene. The ihb gene is identical to oppI, a gene that regulates the oppABCDF operon (E. A. Austin, J. C. Andrews, and S. A. Short, Abstr. Mol. Genet. Bacteria Phages, p. 153, 1989). Thus, oppI/ihb encodes a protein that regulates both ilvIH, an operon that is repressed by leucine, and oppABCDF, an operon involved in peptide transport that is induced by leucine. We propose that the designation lrp be used in the future instead of oppI or ihb and that Lrp (leucine-responsive regulatory protein) be used in place of IHB.     

Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.

Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli. One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region. Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending. The results of DNase I footprinting experiments suggest that Lrp binding to this region facilitates the formation of a higher-order nucleoprotein structure. To define more precisely the degree of bending associated with Lrp binding, one or two binding sites were separately cloned into a pBend vector and analyzed. Lrp induced a bend of approximately 52 degrees upon binding to a single binding site, and the angle of bending is increased to at least 135 degrees when Lrp binds to two adjacent sites. Lrp-induced DNA bending, and a natural sequence-directed bend that exists within ilvIH DNA, may be architectural elements that facilitate the assembly of a nucleoprotein complex.