Interaction of the trp repressor from Escherichia coli with a constitutive trp operator.
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ABSTRACT:
SUBMITTER: Chandler LR
PROVIDER: S-EPMC1148945 | biostudies-other | 1988 Mar
REPOSITORIES: biostudies-other
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Project description:The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all. Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding. Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove. However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove. This negative potential is significantly neutralized in the protein conformation that is bound to DNA. Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change. This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex. The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants. A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies.
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Project description:Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA [for tryptophan (W) repressor-binding protein] remained associated with its namesake through several sequential protein fractionation steps. The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons). The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle.
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Project description:The E. coli trp repressor (trpR) homodimer recognizes its palindromic DNA binding site through a pair of flexible helix-turn-helix (HTH) motifs displayed on an intertwined helical core. Flexible N-terminal arms mediate association between dimers bound to tandem DNA sites. The 2.5 A X-ray structure of trpR crystallized in 30% (v/v) isopropanol reveals a substantial conformational rearrangement of HTH motifs and N-terminal arms, with the protein appearing in the unusual form of an ordered 3D domain-swapped supramolecular array. Small angle X-ray scattering measurements show that the self-association properties of trpR in solution are fundamentally altered by isopropanol.
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Project description:The Escherichia coli K-12 mtr gene, which encodes a tryptophan-specific permease, was cloned, and its nucleotide sequence was determined. The precise location of the mtr gene at 69 min on the E. coli chromosome was determined. The mtr gene product was identified as a 414-amino-acid residue protein with a calculated molecular weight of 44,332. The protein is very hydrophobic, consistent with its presumed location spanning the cytoplasmic membrane. The initiation sites of transcription and translation were identified. Construction of an mtr-lacZ transcriptional fusion facilitated investigation of the molecular basis of mtr regulation. The TyrR protein in association with phenylalanine or tyrosine is responsible for the activation of mtr expression, whereas the Trp repressor in conjunction with tryptophan serves to repress expression of this gene. Site-directed mutagenesis confirmed that sequences in the mtr regulatory region homologous to TyrR protein and to Trp repressor-binding sites were involved in the activation and repression of mtr expression, respectively. Sequences homologous to sigma 70- and sigma 54-dependent promoters were identified upstream of the transcription start point of mtr. It was determined that transcription of mtr occurs only via a sigma 70-dependent promoter.
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Project description:Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)-lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO.
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Project description:The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.
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Project description:The Escherichia coli AraC protein represses and induces the araBAD operon in response to the absence or presence of l-arabinose. Constitutive mutations in the AraC gene no longer require the presence of l-arabinose to convert AraC from its repressing to its inducing state. Such mutations were isolated directly by virtue of their constitutivity or by their resistance to the nonmetabolizable arabinose analog, d-fucose. The majority of the constitutive mutations lie within the same residues of the N-terminal regulatory arm of AraC. Two, however, were found in the core of the dimerization domain. As predicted by the light switch mechanism of AraC, constitutive mutations increase the susceptibility of the N-terminal arms to digestion by trypsin or chymotrypsin, suggesting that these mutations weaken or disrupt the arm structure required for repression by AraC. Fluorescence, circular dichroism, and cysteine reactivity measurements show that the constitutive mutations in the core of the dimerization domain lead to a weakening of the support for the arms and reduce the stability of the minus-arabinose arm structure. These mutations also weaken the interaction between the two-helix bundle and the beta-barrel subdomains of the dimerization domain and reduce the structural stability of the beta-barrels.
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