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1.
In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator sites on Escherichia coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli DNA. In a partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to either non-operator sites on E. coli DNA or to an Oc operator, the formation of complexes between X86 repressor and wild-type operator is favored by low inducer concentrations. Repression of the lac enzymes increases drastically in the X86 mutant in the absence of DNA synthesis in vivo. A new explanation for the in vivo characteristics of the X86 mutant is suggested.  相似文献   

2.
A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (Itb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. Itb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which Itb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.  相似文献   

3.
Summary We have constructed and tested three lac diploid strains in an attempt to show whether operator-constitutive mutations relieve catabolite repression of the lac operon. Each of these carries a different operator mutation on the chromosome, and all three have the genotype I+P+OcZ+Y-polar/Flac I+P+O+ZdelY+A+. When these strains were grown in medium containing glucose plus gluconate, synthesis of -galactosidase (directed by a gene cis to a mutant operator) and of thiogalactoside transacetylase (directed by a gene cis to an intact operator) suffered equal catabolite repression. We conclude that the operator-constitutive mutations have no effect on catabolite repression. Since it has been shown in analogous experiments that all promoter mutations tested do alleviate catabolite repression, these results are consistent with the view that the operator and promoter are functionally distinct.  相似文献   

4.
Hypoxanthine was substituted for guanine at specific sites in the lac operator DNA by a combination of chemical and enzymatic procedures. The stability of these modified lac operators with wild-type (SQ) and tight binding (QX86) lac repressors was measured. Effects were variable. At some sites insertion of hypoxanthine significantly reduced the stability of the complex whereas at other sites substitution with hypoxanthine did not alter the repressor—operator interaction. In addition, insertion of this analog at two sites increased the stability of the complexes. These changes were used to partially map regions of the lac operator that are in contact with lac represser. The results suggest that lac repressor recognizes the guanine 2-amino group at specific sites in the minor groove of lac operator.  相似文献   

5.
The influence of the two operator-like regions lying within or near the lac regulatory region on the binding of lac repressor to lac operator has been investigated. λdlac phages deleted either for the “second operator” in the beginning of the Z gene or deleted for the “third operator” at the end of the I gene were constructed. In in vitro binding experiments it could be shown that the deletion of secondary repressor binding sites from the lac regulatory region does not significantly alter the stability of the repressor—operator complex. Measuring the rate constant of association of repressor with operator in the presence of a 150-fold excess of unspecific DNA, we observed a concentration-dependent effect of the unspecific DNA, although the ratio of operator to non-operator DNA was kept constant. A small effect of the secondary binding sites is seen on the rate of association of repressor with operator, indicating that the secondary binding sites might play a role in facilitating association of repressor with operator under in vivo conditions.  相似文献   

6.
In order to compare the structures of the DNA-binding sites on variants of the lac repressor, we have studied the influence of these variants on the dimethylsulfate methylation of the lac operator. Since a bound protein changes the availability of specific purines in the operator to this chemical attack, comparisons of the methylation patterns will show similarities or differences in the protein DNA contacts. We compared lac repressor, induced lac repressor (repressor bound to the gratuitous inducer isopropyl-β-d-thiogalactoside), mutant repressors having increased operator affinities (X86, I12 and the X86-I12 double mutant) and repressor peptides (long headpiece, residues 1 to 59 and short headpiece. residues 1 to 51). All of these repressors and repressor peptides exhibit the same general pattern of protection and enhancement in the operator; however, the short headpiece pattern differs most from that of the repressor while the induced repressor and the long headpiece show intermediate patterns that are strikingly similar to each other. The mutant repressors do not show an isopropyl-β-d-thiogalactoside effect but otherwise are almost indistinguishable from wild-type repressor. These results demonstrate that all molecules bind to the operator using basically the same protein-DNA contacts; they imply that (1) most and possibly all repressor contacts to operator lie within amino acids 1 to 51, (2) inducer weakens many contacts rather than totally disrupting one or even a few and (3) the tight-binding mutants do not make additional contacts to the DNA.These results are consistent with a model in which the amino-terminal portions of two repressor monomers make the DNA contacts. We show that one can understand the affinity of binding as related to the accuracy of the register of the two amino-terminal portions along the DNA. Furthermore, the action of inducer and the behaviour of the tight binding mutants can be accomodated within a two-state model in which the strongly or weakly binding states correspond to structures in which the amino-terminal regions are rigidly or loosely held with respect to each other.  相似文献   

7.
8.
Sixteen mutants which produce lactose repressors with enhanced operator affinities have been isolated. By deletion mapping, six of seven mutations mapped fall into a restricted region of the i gene which also is the location of some anomalous is (super-repressor) and some weak i?d mutations (Pfahl et al., 1974). In vivo and in vitro characterization of nine of the “tight-binding” repressors indicates that: (1) they cause 1.5- to 6-fold decreases in basal β-galactosidase specific activities relative to the parental Q wild-type repressor, and have up to 30-fold increases in operator affinity in vitro. (2) With a few exceptions, the tight-binding repressors show the same relative decreases in basal β-galactosidase specific activities for a wide range of operator types (o+ and oc). (3) With two exceptions, the tight-binding repressors show normal or nearly normal affinities for the inducer, isopropyl-β-d-thiogalactoside, although the concentrations of inducer needed to release various repressors from the o+ operator vary greatly.  相似文献   

9.
The right operator in bacteriophage lambda vs326 has one-twentieth the in vitro binding affinity for repressor as λv+; for comparison λv3 has one-quarter the affinity of λv+. In vivo, both mutants constitutively express genes in the right operon. Both λv3 and λvs326 express gene O constitutively because they complement λimm434Oam? in a λ lysogen, vs, more efficiently than v3. The v3 allele in cis (but not in trans) to vs326 gives significantly greater phage yields in a λ lysogen than λvs326 alone, cro gene function, measured by arrest of exonuclease synthesis, suggested the following series of increasing degree of conatitutivity: v3, vs326, v3 vs326. λv2 vs326 forms plaques on lysogens that carry λcI857, but λv2 v3 does not. These results indicate that vs326, like v3, is an operator constitutive mutation but stronger in its effects. These mutants exemplify a uniform correlation between relative weakness of repressor binding and degree of constitutive gene expression.  相似文献   

10.
In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.  相似文献   

11.
We have generated more than 300 altered lac repressor proteins carrying known amino acid replacements, by employing nonsense mutations at 90 positions in the lacI gene together with eight different nonsense suppressors. This allows the substitution of lysine, serine, tyrosine, leucine and glutamine at virtually all of the respective positions in the repressor, and tryptophan at ten positions in the repressor. Since most of the nonsense sites have been correlated with specific codons in the lacI messenger RNA, in almost all cases the position of the substituted residue is known. This process results in the creation of a large number of mutant phenotypes. We have analyzed the effects of each substitution in vivo, and in several cases studied partially purified repressors in vitro. The properties of the altered proteins have been compared with the position and nature of each exchanged residue. We discuss the implications of these findings with regard to repressor structure in particular, and to protein structure in general. Further applications of the suppression method are also considered.  相似文献   

12.
The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3′ boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3′ boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.  相似文献   

13.
This paper shows that 19F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule.  相似文献   

14.
The reversion behavior of pleiotropic carbohydrate mutants, previously designated as ctr, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac+ reversions were found in the lac region (11 min) and some Mal+ reversions occurred at malB (79 min), at a distance from the site of the ctr mutations (46 to 47 min). About one-third of Lac+ and Mal+ revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a ctr mutation in a lac constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like ctr mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of ctr were still found to lack enzyme I.  相似文献   

15.
The replication defective transducing phage λplac5O29P3 carries a portion of the E.colilac operon in the b2 region of the lambda phage. This lac operon segment contains the lac promoter, the lac operator, and the β-galactosidase z gene, but does not contain the lac repressor i gene. The z gene can be expressed from both the inserted lac promoter and the phage promoter. When E.coli strain 594 (z?, i+) or JC6256 (Δlac) is infected by λplac5O29P3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ+) or JC6256 (λ+) is infected by λplac5O29P3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted lac promoter.The ability to separate the phage promoter from the inserted lac promoter for β-galactosidase expression will simplify the interpretation whenever λplac5 is used.  相似文献   

16.
17.
The interaction oflacoperator DNA withlacrepressor (LacI) is a classic example of a genetic regulatory switch. To dissect the role of stoichiometry, subunit association, and effects of DNA length in positioning this switch, we have determined binding isotherms for the interaction of LacI with a high affinity (Osym) operator on linearized plasmid (2500 bp) DNA over a wide range of macromolecular concentrations (10−14to 10−8M). Binding data were analyzed using a thermodynamic model involving four equilibria: dissociation of tetramers (T) into dimers (D), and binding of operator-containing plasmid DNA (O) to dimers and tetramers to form three distinct complexes, DO, TO, and TO2. Over the range of con- centrations of repressor, operator, and salt (0.075 M K+to 0.40 M K+) investigated, we find no evidence for any significant thermodynamic effect of LacI dimers. Instead, all isotherms can be interpreted in terms of just two equilibria, involving only T and the TO and TO2complexes. As a reference binding equilibrium, which we propose must approximate the DO binding interaction, we compare the plasmid Osymresults with our extensive studies of the binding of a 40 bp OsymDNA fragment to LacI. On this basis, we obtain a lower bound on the LacI dimer – tetramer equilibrium constant and values of the equilibrium constants for formation of TO and TO2complexes.At a salt concentration of 0.40 M, the Osymplasmid binding data are consistent with a model with two independent and identical binding sites for operator per LacI tetramer, in which the binding to a site on the tetramer is only slightly more favorable than the reference binding interaction. Increasingly large deviations from the independent-site model are observed as the salt concentration is reduced; binding of a second operator to form TO2becomes strongly disfavored relative to formation of TO at low salt concentrations (0.075 to 0.125 M). In addition, binding of both the first and second plasmid operator DNA molecules to the tetramer becomes increasingly more favorable than the reference binding interaction as [K+] is reduced from 0.40 M to 0.125 M. At 0.075 M K+, however, the strength of binding of the second plasmid operator DNA to the LacI tetramer is dramatically reduced; this interaction is much less favorable than binding the first plasmid operator DNA, and becomes much less favorable than the reference binding interaction. We propose that these differences arise from changes in the nature of the TO and TO2complexes with decreasing salt concentration. At low salt concentration, we suggest the hypothesis that flanking non-operator sequences bind non-specifically (coulombically) by local wrapping, and that distant regions of non-operator DNA occupy the second operator-binding site by looping. We propose that wrapping stabilizes both 1:1 and 2:1 complexes at low salt concentration, and that looping stabilizes the 1:1 complex but competitively destabilizes the 2:1 TO2complex at low salt concentration. These effects must play a role in adjusting the stability and structure of the LacI-lac operator repression complex as the cytoplasmic [K+] varies in response to changes in extracellular osmolarity.  相似文献   

18.
Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.  相似文献   

19.
Ion concentrations (K+, Glu) in the cytoplasm of growingEscherichia colicells increase strongly with increases in the osmolarity of a defined growth medium. Whilein vitroexperiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration,in vivomeasurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations ofin vivototal concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, andin vitrobinding data on the effects of putrescine concentration on a specific PNAI (lacrepressor-lacoperator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (∼64 mmol (l H2O)−1at 0.03 OsM) to high osmolarity (∼8 mmol (l H2O)−1at 1.02 OsM). Over this osmotic range the total [K+] increases from ∼0.2 mol (l H2O)−1to ∼0.8 mol (l H2O)−1. We find that the effect of putrescine concentration on the repressor-operator interactionin vitrois purely competitive and is quantitatively described by a simple competition formalism in whichlacrepressor behaves as a specific-binding oligocation (ZR=8±3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+concentration in response to changes in external osmolarity provide a general mechanism forE. colito vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.  相似文献   

20.
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