Lac repressor-operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA.
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.
Artz
P G
PG
Department of Chemistry, Albright College, Reading, PA 19612-5234, USA.
Valentine
K G
KG
Opella
S J
SJ
Lu
P
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007763:Lac Operon; D056968:Lac Repressors; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009875:Operator Regions, Genetic; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012097:Repressor Proteins