DNA–protein interaction in solution: NMR studies of the <sup>13</sup>C‐site‐specifically labelled Lac operator–Lac repressor complex

Lancelot, G.; Maurizot, Jean‐Claude; Chassignol, Marcel; Thuong, Nguyen T.; Chanteloup, Luc; Beau, Jean‐Marie

doi:10.1002/mrc.1260321007

Cited by 3 publications

(1 citation statement)

References 12 publications

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“…1). The assignment of the 1 H and 13 C resonances has been published previously 17 and was used here. The experimental relaxation rates R(C z ), R(C x,y ) and R(H z !…”

Section: Relaxation Rate Datamentioning

confidence: 99%

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

2000

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Structural and dynamic studies of the lac Operator complexed with the headpiece of the lac Repressor are necessary to establish whether the two partners have pre‐required structures or if folding is essential to induce the specific interaction. NOESY spectra of the 1 : 1 complex of half of the lac Operator 5′d(CGCTCACAATT)–5′d(AATTGTGAGCG) sequence (selectively labelled with 13C at C‐1′), with the lac Repressor N‐terminal headpiece 1–51 yielded information about the contact points between the nucleic acid and the protein. The operator was selectively labelled with 13C at the C‐1′ position, allowing the measurement of 13C relaxation rates as a probe of dynamic behaviour. Measurement of the relaxation rates of R(Cz), R(Cx,y) and R(Hz → Cz) within the complex were carried out and a sequence dependence of R(Cx,y) was observed. The different possible relaxation processes were analysed and the data were interpreted in terms of chemical exchange between two conformations of the operator. It is concluded that the complexation of the N‐terminal headpiece 1–51 with the lac Operator induced conformational changes of the nucleic acid. These data, in combination with reported 15N relaxation rates, suggest that the mobility of some regions of the nucleic acid and of the protein are essential for effective interaction between the two molecules. Copyright © 2000 John Wiley & Sons, Ltd.

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“…1). The assignment of the 1 H and 13 C resonances has been published previously 17 and was used here. The experimental relaxation rates R(C z ), R(C x,y ) and R(H z !…”

Section: Relaxation Rate Datamentioning

confidence: 99%

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

2000

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Lac repressor–operator interaction: N-terminal peptide backbone1H and15N chemical shifts upon complex formation with DNA

et al. 1996

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When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contracted asymmetrically. This asymmetric binding results from the DNA sequence/structure. The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone. We isolated uniformly 15N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant [Pro3 > Tyr3] (HP64tyr3) lac repressor N-terminal DNA binding fragments for 1H/15N NMR studies with the left and right operators separately. Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances. For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar. Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar. On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator. Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.

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NMR Studies of lac Operator and lac Repressor

Lancelot

Paquet

2003

Annual Reports on NMR Spectroscopy

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DNA–protein interaction in solution: NMR studies of the ¹³C‐site‐specifically labelled Lac operator–Lac repressor complex

Cited by 3 publications

References 12 publications

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

Lac repressor–operator interaction: N-terminal peptide backbone1H and15N chemical shifts upon complex formation with DNA

NMR Studies of lac Operator and lac Repressor

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DNA–protein interaction in solution: NMR studies of the 13C‐site‐specifically labelled Lac operator–Lac repressor complex

Cited by 3 publications

References 12 publications

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

Changes inlac Operator dynamics upon selective interaction withlac Repressor as revealed by heteronuclear relaxation rate measurements

Lac repressor–operator interaction: N-terminal peptide backbone1H and15N chemical shifts upon complex formation with DNA

NMR Studies of lac Operator and lac Repressor

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Product

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About

DNA–protein interaction in solution: NMR studies of the ¹³C‐site‐specifically labelled Lac operator–Lac repressor complex