Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Schnarr, Manfred; Durand, M.; Maurizot, Jean‐Claude

doi:10.1021/bi00284a005

Cited by 13 publications

(22 citation statements)

References 63 publications

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“…2). However, our results, those of Winter and von Hippel [9] and those of Schnarr et al [34] span a greater range of salt concentrations and extend to lower salt concentrations (below 50 mM)…”

Section: 'supporting

confidence: 74%

Analysis of the Thermodynamic Linkage of DNA Binding and Ion Binding for Dimeric and Tetrameric forms of the Lac Repressor

Stickle

Liu

Fried

1994

European Journal of Biochemistry

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The salt concentration dependences of the observed association constants (Kobe) for the binding of wild-type lac repressor tetramer and the dimeric lad-18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion-binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion-binding affinities requires additional mechanistic or structural information. The simplest ion-binding mechanism consistent with the data is compatible with a current structural model of the repressor-operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild-type repressor binding is slightly greater than that of the lad-18 mutant. This difference may reflect the availability of ion-binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.The observed equilibrium association constant (Kobs) for protein-nucleic-acid interactions is strongly dependent on the concentration and identity of ions present in the surrounding solution. The thermodynamics of the influence of ions on protein -nucleic-acid interactions has been described by If ion-binding reactions of protein and DNA are the only significant contributors to the influence of the concentration of a single monovalent salt (MX) on Koba, then the following relationship is predicted P I :where Am = the number of cations gained by the protein upon binding, An = the number of anions gained by the protein upon binding, and Aq = the number of cations gained by nucleic acid upon binding. [MX]. One explanation that is consistent with this observation is that it is due to the displacement, by protein binding, of cations from the negatively-charged nucleic acid surface ( A q < 0), and that ion-binding by the protein does not contribute (Am = An = 0). Since the cation concentration near the nucleic acid surface is almost independent of the bulk salt concentration for 10 mM d [MX]

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Section: 'supporting

confidence: 74%

Analysis of the Thermodynamic Linkage of DNA Binding and Ion Binding for Dimeric and Tetrameric forms of the Lac Repressor

Stickle

Liu

Fried

1994

European Journal of Biochemistry

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“…This conformational change is very similar in both cases to that induced by the entire lac repressor or its amino-terminal headpiece (Maurizot et al, 1974;Butler Fig. et Schnarr et al, 1983). Consensus protection patterns for different operators produced by LexA and bacteriophage X repressor.…”

Section: Discussionmentioning

confidence: 66%

“…The centre of symmetry of the operators is indicated by a vertical slash. et Schnarr et al, 1983). The c.d.…”

Section: Discussionmentioning

confidence: 99%

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Hurstel¹,

Granger-Schnarr²,

Daune³

et al. 1986

The EMBO Journal

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Both the amino‐terminal and the carboxy‐terminal domain of the LexA repressor have been purified using the LexA protein autodigestion reaction at alkaline pH, which leads to the same specific products as the physiological RecA‐catalyzed proteolysis of repressor. We show by circular dichroism (c.d) that, upon non‐specific binding to DNA, the purified amino‐terminal domain induces a very similar if not identical conformational change of the DNA as does the entire repressor. The positive c.d. signal increases approximately 3‐fold if the DNA lattice is fully saturated with protein. Further, the amino‐terminal domain of the LexA protein binds specifically to the operator of the recA gene, producing qualitatively the same effects on the methylation pattern of the guanine bases by dimethylsulfate as the entire repressor, consisting of a methylation inhibition effect at four distal operator guanines and a slight enhancement at the central bases. The spacing between these contacts suggests that LexA does not bind to the operator along the same face of the DNA helix. As shown by c.d. studies the amino‐terminal domain harbours a substantial amount of residues in alpha‐helical conformation, a prerequisite for DNA recognition via a helix‐‐turn‐‐helix structural motif as proposed for many other regulatory proteins.

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“…After Sepharose 6B chromatography of the irradiated complex, the presence of the headpiece in the DNA peak was assayed by fluorescence measurements. Corrections for the screening effect, and for quenching by DNA binding (Schnarr et al, 1983), were made. Nevertheless, irradiations performed under the same conditions as for the repressor do not induce any cross-link.…”

Section: Resultsmentioning

confidence: 99%

“…Considering that the binding of isolated headpieces induces similar changes in the CD spectrum of the DNA as the binding of intact repressor (Schnarr et al, 1983), it is highly probable that the conformational changes are in both cases quite similar.…”

Section: Discussionmentioning

confidence: 99%

Photochemical crosslinking of lac repressor to nonoperator 5-bromouracil-substituted DNA

Barbier¹,

Charlier²,

Maurizot³

1984

Biochemistry

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Ultraviolet irradiation of lac repressor bound to 5-bromouracil-substituted nonoperator DNA leads to the formation of cross-links between the protein and the nucleic acid. The cross-links are formed between the DNA and the 1-51 N-terminal peptide of the repressor, the "headpiece". The tetrameric core (4 X 60-360 amino acids) was never found to be cross-linked to the DNA. With isolated headpieces, which are able to bind DNA, no cross-link was detected. These results are discussed considering the fundamental role of the core in keeping the headpieces in an adequate geometry for the DNA-repressor interaction. It has been possible to cross-link two DNA molecules to one repressor molecule, thus showing the existence of at least two binding sites for nonoperator DNA on the repressor. The attached peptides were analyzed after extensive proteolytic cleavage, and the most abundant peptide found was peptide 23-33. Histidine-29 seems to be the photo-cross-linked amino acid. Analysis of the results required a computation method discussed in the Appendix.

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Nonspecific interaction of the lac repressor headpiece with deoxyribonucleic acid: fluorescence and circular dichroism studies

Cited by 13 publications

References 63 publications

Analysis of the Thermodynamic Linkage of DNA Binding and Ion Binding for Dimeric and Tetrameric forms of the Lac Repressor

Analysis of the Thermodynamic Linkage of DNA Binding and Ion Binding for Dimeric and Tetrameric forms of the Lac Repressor

In vitro binding of LexA repressor to DNA: evidence for the involvement of the amino-terminal domain.

Photochemical crosslinking of lac repressor to nonoperator 5-bromouracil-substituted DNA

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