Association of Escherichia coli lac repressor with poly[d(A-T)] monitored with 8-anilino-1-naphthalenesulfonate

Worah, Dilip M.; Gibboney, Kathleen M.; Yang, Lian-Mei; York, Sheldon S.

doi:10.1021/bi00614a020

Cited by 15 publications

(20 citation statements)

References 28 publications

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“…The data presented in Figures 5 and 6 suggest that bis-ANS and nucleic acid bind to the same region of the NC protein. This is supported by reports that bis-ANS has a particular affinity for proteins that have nucleotide binding sites (Worah et al, 1978;Wu & Wu, 1978;Prasad et al, 1986;Lin et al, 1983;Holler et al, 1971). The tight binding between the NC protein and poly (A), as measured by the displacement of bis-ANS, is consistent with previous observations, as is the site size of 6 (Jentoft et al, 1988).…”

Section: Discussionsupporting

confidence: 89%

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Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

Secnik¹,

Wang²,

Chang³

et al. 1990

Biochemistry

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The structural and functional properties of the nucleocapsid (NC) protein of the avian myeloblastosis virus were examined by steady-state fluorescence and fluorescence anisotropy measurements of the complex between the NC and the extrinsic fluorophore 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid (bis-ANS). The intrinsic fluorescence of bis-ANS is enhanced many fold upon forming a complex with the NC. Between 2 and 10 molecules of bis-ANS bind strongly to the NC, with an overall Kd of less than 10(-6) M. The emission of bis-ANS in the complex can also be induced by excitation at 298 nm, indicating that energy is transferred from Trp 80, the sole tryptophan in the NC protein, to bis-ANS. The energy transferred between the Trp 80 and bis-ANS was analyzed to yield a calculated distance of separation between these fluorophores of 28 +/- 3 A; thus, Trp 80 is well removed from the nearest bound bis-ANS. The fluorescence emission of bis-ANS in the NC.bis-ANS complex is efficiently quenched by added salts and by poly(A), suggesting that salt (presumably anions), nucleic acid, and bis-ANS bind to the same, positively charged region on the NC protein. A site size of six nucleotides was determined for nucleic acid binding to the NC protein, with an estimated Kd of less than 10(-6) M. Salt (anion) binding is strong, but nonspecific, with a Kapp of 4 mM, raising the possibility that anion binding to the NC protein might regulate the interaction of the NC with viral RNA inside the host cell.

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Section: Discussionsupporting

confidence: 89%

“…Multiple binding sites for bis-ANS have been reported for other proteins such as RNA polymerase figure 6: Plot of the quenching of the bis-ANS fluorescence in the NC-bis-ANS complex by poly(A) (representative data shown in Figure 5). (Wu & Wu, 1978), E. coli lac repressor (Worah et al, 1978), and tubulin (Prasad et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

Secnik¹,

Wang²,

Chang³

et al. 1990

Biochemistry

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“…A decrease in the contact between the NH2 terminus and its core as a result of nonspecific DNA binding is suggested by the results of Worah et al (47). In light of these observations, we favor the interpretation that binding of nonspecific DNA to the NH2 terminus results in a dissociation of this region from its contact with the core protein (thus resulting in release of a portion of the anilinonaphthalenesulfonate molecules bound at that interface).…”

supporting

confidence: 56%

Model for lactose repressor protein and its interaction with ligands.

Dunaway¹,

Manly²,

Matthews³

1980

Proc. Natl. Acad. Sci. U.S.A.

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A model is presented for the structure of the lactose repressor protein and for its interaction with inducer, operator DNA, and nonspecific DNA. The proposed structure is based on experimental evidence from this laboratory and from the literature and is offered as an integration of the available data on this system. Features unique to this model include: (i) interaction of the core region of the protein with the operator, (ii) primary effects of the conformational change in response to inducer on the core-operator interaction, (iii) contacts between all four subunits of the protein and the operator DNA, and (iv) qualitative differences in operator and nonspecific DNA binding.The lactose repressor protein interacts specifically with the operator sequence in Escherichia coli DNA to prevent transcription of the DNA coding for the lac enzymes (1). This binding is altered by the presence of sugar ligands (inducers), which elicit a conformational change in the protein to a state with lower affinity for the operator DNA sequence (2). The physical dissociation of the repressor protein from the operator region is accomplished through competition of the remainder of the E. coli genome (nonspecific DNA) for binding to the repressor protein; the nonspecific DNA binding affinity is unaffected by inducer binding and is of sufficient magnitude that other DNA competes with operator effectively only in the presence of the sugar ligand (3). The assignment of the NH2 termini as the site for repressor-DNA interactions and the probable involvement of only two subunits of the protein in this process have been assumed in the previous models proposed for repressor binding to DNA (1,(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Specifically, Kania and Brown (13) and more recently Ogata and Gilbert (6) proposed a model for repressor-operator interaction in which only two subunits of the protein bind to operator. According to Ogata and Gilbert (6), this binding occurs via the NH2-terminal domains (amino acids 1-59), which are presumed to contain all determinants for both specific and nonspecific DNA binding (6); the inducer-associated conformational change is transmitted to the NH2-terminal domains from the core protein via a hinge region (amino acids 60-80) which has been previously postulated (6,(14)(15)(16). From a detailed analysis of lacOc mutants, base substitution studies of operator DNA, and correlation of these with operator methylation and crosslinking patterns in the presence of repressor, Goeddel et al. (17) have suggested that the repressor does not lie parallel to the helix axis but crosses the axis at a 10-200 angle (17). Recent data from our laboratory combined with the previous information in the literature provide the basis for postulating a model for the structure of the repressor protein and its interaction with ligands that differs from the models in the literature in several important features.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby mar...

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“…The lac repressor was isolated from E. coli CSH 46 and assayed for operator binding activity as described (13). Other materials were obtained from the following sources:…”

mentioning

confidence: 99%

lac repressor changes conformation upon binding to poly[dA-T)]

Kelsey¹,

Rounds²,

York³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The association of Escherichia coli lac repressor with its operator and with nonoperator DNA has been extensively studied (for recent reviews, see refs. 1-3). Both the lac operator (4) and nonoperator DNA (5, 6) undergo structural changes as a consequence of repressor binding. The large bimolecular rate constant for association (7,8) implies that these changes occur within the repressor-DNA complex. We present evidence that the lac repressor undergoes at least two conformational changes as it binds to poly[d(A-T)]. Quite likely, one or more of these changes occurs simultaneously with changes in the DNA, serving to strengthen their interaction.The lac repressor was treated with N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonate (I-AENS) (9) to covalently label cysteine residues with the fluorescent N-(acetylaminoethyl)-1-naphthylamine-5-sulfonate (AENS) group. Each repressor subunit has three cysteine residues (10) located in regions not thought to be directly involved in DNA binding (3). These residues display different reactivities toward the chromophoric reagents 2-chloromercuri-4-nitrophenol and 2-bromoacetamido-4-nitrophenol (11). However, the fluorescent reagent fluorescein mercuric acetate shows little selectivity among the three sulfhydryls (12). We find that I-AENS reacts selectively with one cysteine residue in each repressor subunit. Stopped-flow techniques have been used to monitor the events during the association of repressor with poly[d(A-T)] that alter the fluorescence of this AENS label.MATERIALS AND METHODS Materials. The lac repressor was isolated from E. coli CSH 46 and assayed for operator binding activity as described (13). Other materials were obtained from the following sources:poly[d(A-T)] from Miles, I-AENS from Aldrich, iodo[14C]-acetamide from New England Nuclear, and trypsin (TRL) and chymotrypsin (CDI) from Worthington.Labeling of lac Repressor. Repressor (1-3 mg/ml) in 0.2 M Tris-HCI (pH 8.6 at 40C), 0.5 M KCI, and 0.1 mM EDTA, was reacted with I-AENS (usually 8.1 mM) at 40C in the dark. At the appropriate time, the protein was separated from excess reagent by gel filtration through Sephadex G-25 and then dialyzed overnight against the buffer used for labeling, with 0.1 mM dithiothreitol added. The concentration of repressor and extent of AENS labeling were determined from the absorbance at 280 and 340 nm by using the following values: lac repressor subunit, E2W = 22,125 M-1 cm-1 (14), Ec40 = 105 M-1 cm-1; AENS, E280 = 1260 M-1 cm-1, E3O = 6850 M-1 cm-1 (9).Identification of Labeled Cysteine Residues. AENS-labeled repressor was reacted with iodo[14C]acetamide (1.68 Ci/mole, 1 Ci = 3.70 X 1010 becquerels) in 8 M urea at 320C. The molar concentration of iodoacetamide was 6 times that of repressor subunits. After 30 min, excess iodoacetamide was reacted with dithiothreitol, and the splution was dialyzed against 0.1 M NH4HCO3/2 M urea. This protein was then digested with trypsin (1% wt/wt) for 2.5 hr at 250C. A second addition of trypsin was followed by an additional 2.5-hr digestion....

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Association of Escherichia coli lac repressor with poly[d(A-T)] monitored with 8-anilino-1-naphthalenesulfonate

Cited by 15 publications

References 28 publications

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

Interactions at the nucleic acid binding site of the avian retroviral nucleocapsid protein: studies utilizing the fluorescent probe 4,4'-bis(phenylamino)(1,1'-binaphthalene)-5,5'-disulfonic acid

Model for lactose repressor protein and its interaction with ligands.

lac repressor changes conformation upon binding to poly[dA-T)]

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