Leucine-responsive Regulatory Protein-DNA Interactions in the Leader Region of the ilvGMEDA Operon of Escherichia coli

Abstract

The leucine-responsive regulatory protein, Lrp, 1 is a global regulatory protein of Escherichia coli that affects the expression of many operons (reviewed in Refs. 1 and 2). The expression of some target operons is activated by Lrp, while the expression of others is repressed. In addition, the free amino acid, L-leucine, acts as an effector ligand of Lrp; and, at some DNA target sites, L-leucine is required for Lrp binding. At other sites, L-leucine can antagonize or have no effect on Lrp-DNA interactions. In most cases, the physiological role of Lrp is to activate biosynthetic operons and to repress degradative ones. Therefore, it has been suggested that Lrp and L-leucine might function to coordinate metabolic shifts between nutritional feast and famine conditions. However, the metabolic coherence of the operons regulated by Lrp remains unclear.The structural gene for Lrp was initially identified as a mutation in a regulatory gene, livR, for the branched-chain amino acid (L-leucine, L-isoleucine, and L-valine) transport system (3). Subsequently, additional mutations in this gene that affected the biosynthesis of L-leucine were identified (4). Further studies showed that the protein product of this gene (now referred to as lrp) activates transcription of the ilvIH operon, which encodes one of the three acetohydroxy acid synthase (AHAS) isozymes required for the first step in the biosynthesis of the three branched-chain amino acids (4). Interestingly, many of the other operons regulated by Lrp are involved in either the production or the dissimilation of the carbon substrates, pyruvate and ␣-ketobutyrate, of the AHAS isozymes required for branched-chain amino acid biosynthesis (Fig. 1). For example, Lrp regulates the serA and sdaA genes involved in the production of pyruvate and the tdh and kbl genes, which affect the in vivo levels of L-threonine and ␣-ketobutyrate (5, 6). Also, Lrp regulates the expression of the leucine biosynthetic operon, leuABCD (7), and the expression of the branched-chain amino acid transport genes livJ and livKHMG (8). Thus, it appears that Lrp plays an important role in the maintenance of appropriate in vivo levels of the branched-chain amino acids. Such a role for Lrp in branched-chain amino acid metabolism is further underscored by the partial auxotrophy for the branched-chain amino acids observed in a lrp Ϫ strain of E. coli K12 (1).In E. coli, the biosynthesis of the branched-chain amino acids occurs via a parallel pathway catalyzed by several bifunctional enzymes ( Fig. 1) (9). The genes encoding these enzymes are specified by four separate operons (ilvBN, ilvGMEDA, ilvYC, and ilvIH). The ilvBN and ilvIH operons encode the genes for the subunits of the AHAS I and AHAS III isozymes, respectively (10). The ilvGMEDA operon encodes four of the five enzymes required for the biosynthesis of L-isoleucine and Lvaline (11). The ilvGM genes encode the subunits of AHAS I, while the remaining genes encode two other enzymes of the common pathway, dihydroxy-acid dehydratase (ilvD) and transamin...

Global Gene Expression Profiling in Escherichia coliK12

“…We have previously used an ilvP G ::lacZ construct to measure ␤-galactosidase activities in the same isogenic lrp ϩ and lrp Ϫ strains employed in this study (7). These results showed that ␤-galactosidase was increased 2.5-fold in the lrp Ϫ mutant strain.…”

“…Bacteria Strains and Growth Conditions-Strain IH-G2490 (ilvP G ::lacZYA) was constructed by ligating a 515-bp EcoRI-BamHI DNA fragment containing a 494-bp ilvGMEDA-derived HinFI fragment (base pair position Ϫ245 to ϩ249) into the EcoRI and BamHI sites of the lacZ-truncated pRS551⌬ (yielding the reporter plasmid pRSG2490) and integrating this reporter plasmid construct into the bacterial chromosome of the polA-deficient strain, NO3434, as described previously (7). An isogenic lrp derivative of strain IH-G2490 was created by generalized P1 transduction of the lrp-35::Tn10 allele into this strain according to the methods of Miller (8) to yield strain IH-G2491 (ilvP G ::lacZYA, lrp::Tn10).…”

Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

“…The first step to reveal the mechanism of ald regulation was to identify cis-regulatory elements in the upstream region of ald. According to previously reported footprinting results, a long stretch of DNA was generally protected by Lrp/AsnC family regulators (48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61), which indicates the presence of multiple binding sites on DNA. Likewise, four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N 2 -WWN/NWW-N 2 -T/AC were identified upstream of the ald gene by sequence analysis in conjunction with DNase I footprinting analysis and EMSAs.…”

Regulation of Pap Phase Variation

“…Lrp could repress transcription by alternative mechanisms such as interaction with transcriptional regulators or RNA polymerase. For example, work by Rhee et al (33) showed binding of Lrp to the leader-attenuator region of ilvGMEDA 226-bp downstream of the transcription start site is necessary for repression. Repression of ilvGMEDA occurred at the same concentration of Lrp (16 nM) as repression of papBA transcription (Fig.…”