Lac Repressor

Platt, Terry; Files, James G.; Weber, Klaus

doi:10.1016/s0021-9258(19)44452-9

Cited by 215 publications

(52 citation statements)

References 37 publications

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“…Truncated forms of both of these proteins, in which the DNA binding domain has been deleted, have been studied with respect to effector binding. The affinity of the lac repressor core domain for IPTG is indistinguishable from that of the intact repressor for effector (Platt et al, 1973). The N-terminal domain of CRP obtained from chymotrypsincatalyzed cleavage of the intact protein is altered in its ability to bind cyclic AMP (Heyduk et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The tryptophan repressor is an example of a protein in which the effector or corepressor is intimately involved in the sequence-specific DNA binding interaction and is found at the protein-DNA interface. In both the lac repressor and CRP, the effector binding sites are physically distinct from the DNA binding domains (Platt et al, 1973;Eilen et al, 1978;Aiba & Krakow, 1981). Truncated forms of both of these proteins, in which the DNA binding domain has been deleted, have been studied with respect to effector binding.…”

Section: Discussionmentioning

confidence: 99%

“…A combination of biochemical and genetic evidence, as well as high-resolution structural data, indicates that many of the regulatory proteins that respond to effector binding are modular in structure. DNA and effector binding functions are found in separate globular domains in several of these proteins including the Escherichia coli lac repressor (Platt et al, 1973) and the cAMP receptor protein (CRP) (Eilen et al, 1978;Aiba & Krakow, 1981;McKay et al, 1982). Since the affinities of these proteins for their target sites on DNA are modulated by binding of small molecule effectors, interaction between the structural domains within these proteins is central to their function.…”

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confidence: 99%

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Evidence for Interdomain Interaction in the Escherichia coli Repressor of Biotin Biosynthesis from Studies of an N-Terminal Domain Deletion Mutant

Beckett

1996

Biochemistry

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The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA binding protein. The protein is composed of three structural domains. Contact with the biotin operator (bioO) in the transcriptional repression complex is made by the N-terminal domain which contains a helix-turn-helix structural module. The central domain is required for the catalytic functions of BirA including synthesis of biotinyl-5'-AMP from substrates ATP and transfer of biotin from the adenylate to a lysine residue of the biotin carboxyl carrier protein (BCCP) of acetyl CoA carboxylase. The adenylate serves not only as the activated intermediate in the biotin transfer reaction but also as the positive allosteric effector for site-specific DNA binding. Little interaction between the N-terminal and central domains is observed in the apo-repressor structure (Wilson et al., 1992). In this work, we have engineered an N-terminal deletion mutant of BirA, BirA65-321. Biochemical analysis of the purified truncated repressor indicates that, as expected, it does not bind to biotin operator DNA. BirA65-321 is, moreover, identical to intact BirA in catalysis of synthesis of bio-5'-AMP and in transfer of biotin from the adenylate to BCCP. Deletion of the DNA binding domain severely compromises the ability of BirA to bind to biotin or bio-5'-AMP. The affinity of BirA65-321 for biotin is decreased 100-fold while that for bio-5'-AMP is decreased 1000-fold, relative to intact BirA. The significant functional role of the DNA binding domain in tight binding of the two ligands to the central domain may be indicative of formation of extensive interdomain contacts in the holorepressor structure.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Evidence for Interdomain Interaction in the Escherichia coli Repressor of Biotin Biosynthesis from Studies of an N-Terminal Domain Deletion Mutant

Beckett

1996

Biochemistry

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“…Extensive genetic and chemical studies have provided insight into the correlation between primary structure of this protein and its multiple functional activities (Miiller-Hill, 1975;Schmitz etal., 1976;Miller, 1979;Miller et al, 1979;Gordon et al, 1988;LeClerc et al, 1988; Kleina & Miller, 1990). The protein generally can be dissected into two principal structural domains: the N-terminal 59 residues contain a helix-turn-helix motif that confers most of the DN A binding capacity (Adler et al, 1972;Platt etal., 1973;Lin & Riggs, 1975;Lamerichset al, 1989), and the remaining core protein contains the inducer binding site and assembly determinants (Platt et al, 1973; Miiller-Hill, 1975; Schmitz et al, 1976;Miller, 1979;Miller et al, 1979; Kleina & Miller, 1990; Alberti et al, 1991;Chen & Matthews, 1992a). The region that encompasses Tyr282 appears to be essential for assembly of the protein into a tetrameric species, as substitution by amino acids other than Leu or Phe results in a monomeric product t This work was supported by grants from the National Institutes of Health (GM 22441 to K.S.M.…”

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confidence: 99%

Construction of A Dimeric Repressor: Dissection of Subunit Interfaces in Lac Repressor

et al. 1994

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Formation of the lactose repressor tetramer is postulated to involve two subunit interfaces, one primarily contributing to monomer-monomer assembly to dimer and the second to dimer-dimer association to tetramer. The latter interface requires a heptad repeat of three leucines at the C-terminus of lac repressor that is presumed to form an abbreviated coiled-coil motif [Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., & Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Krämer, H., & Müller-Hill, B. (1991) New Biol. 3, 57-62; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. To strengthen the dimer-dimer interface, this motif was extended by the addition of one and two leucine heptad repeat units to the C-terminus by site-specific insertion mutagenesis. The tetrameric products displayed operator and inducer affinity essentially indistinguishable from the wild-type repressor. In order to probe the effect of the elongated coiled-coil on assembly of the repressor tetramer, the other of the two postulated subunit interfaces was disrupted by introducing a point mutation (Y282D) that yields a monomeric protein in the wild-type background. Both elongated mutant repressors were able to assemble into dimeric species, apparently due to the strengthened subunit association at the C-terminal region compared to the wild-type repressor. These results further confirm the role of a coiled-coil structure in the formation of tetramer in the lac repressor.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Trypsin Digestion. Mild trypsin digestion of wild-type lac repressor results in major polypeptides of ~7 and ~20-30 kDa in molecular mass (Platt et al, 1973). In a 12.5% SDS gel, as shown in Figure 8, only the larger polypeptides are visible by silver staining.…”

Section: Resultsmentioning

confidence: 96%

Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor

Chang¹,

Barrera²,

Matthews³

1994

Biochemistry

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To explore the roles of three aspartate residues, Asp88, Asp130, and Asp274, found in the proposed inducer binding site of lac repressor [Sams, C. F., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1984) Nature 310, 429-430], each site was substituted with alanine, glutamate, lysine, or asparagine by site-specific mutagenesis. The mutations at the Asp88 site resulted in a 5-13-fold decrease in inducer binding affinity, largely due to an increase in the inducer dissociation rate constants for these mutants. In addition, the mutant proteins Asp88-->Ala and Asp88-->Lys exhibited altered allosteric behavior for inducer binding. These data conflict with the original hypothesis placing Asp88 in the inducer binding site, but are in agreement with a recent model that places this amino acid close to the subunit interface involved in cooperativity associated with inducer binding [Nichols, J. C., Vyas, N. K., Quiocho, F. A., & Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612; Chen, J., & Matthews, K. S. (1992) J. Biol. Chem. 267, 13843-13850]. Substitution at Asp130 did not alter the inducer binding affinity nor other binding activities. Thus, this amino acid is not crucial in the binding to beta-substituted monosaccharides or in protein function. In stark contrast, all mutant proteins with substitutions at the Asp274 site exhibited no detectable inducer binding. With the exception of Asp274-->Lys, the structures of these mutant proteins appear to be similar to wild-type. The data demonstrate that Asp274 plays a crucial role in inducer binding of this transcriptional regulator.

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Lac Repressor

Cited by 215 publications

References 37 publications

Evidence for Interdomain Interaction in the Escherichia coli Repressor of Biotin Biosynthesis from Studies of an N-Terminal Domain Deletion Mutant

Evidence for Interdomain Interaction in the Escherichia coli Repressor of Biotin Biosynthesis from Studies of an N-Terminal Domain Deletion Mutant

Construction of A Dimeric Repressor: Dissection of Subunit Interfaces in Lac Repressor

Identification and characterization of aspartate residues that play key roles in the allosteric regulation of a transcription factor: aspartate 274 is essential for inducer binding in lac repressor

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