title: Operator DNA sequence variation enhances high affinity binding by hinge helix mutants of lactose repressor protein.
abstract: The mechanism by which genetic regulatory proteins discern specific target DNA sequences remains a major area of inquiry. To explore in more detail the interplay between DNA and protein sequence, we have examined binding of variant lac operator DNA sequences to a series of mutant lactose repressor proteins (LacI). These proteins were altered in the C-terminus of the hinge region that links the N-terminal DNA binding and core sugar binding domains. Variant operators differed from the wild-type operator, O(1), in spacing and/or symmetry of the half-sites that contact the LacI N-terminal DNA binding domain. Binding of wild-type and mutant proteins was affected differentially by variations in operator sequence and symmetry. While the mutant series exhibits a 10(4)-fold range in binding affinity for O(1) operator, only a approximately 20-fold difference in affinity is observed for a completely symmetric operator, O(sym), used widely in studies of the LacI protein. Further, DNA sequence influenced allosteric response for these proteins. Binding of this LacI mutant series to other variant operator DNA sequences indicated the importance of symmetry-related bases, spacing, and the central base pair sequence in high affinity complex formation. Conformational flexibility in the DNA and other aspects of the structure influenced by the sequence may establish the binding environment for protein and determine both affinity and potential for allostery.
authors.affiliation: Department of Biochemistry & Cell Biology, Rice University, Houston, Texas 77251, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Falcon
authors.forename: K S
authors.initials: KS
authors.lastname: Matthews
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020029:Base Pairing / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D014644:Genetic Variation / Q000235:genetics*; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D056968:Lac Repressors; D007785:Lactose / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D007785:Lactose; D005998:Glycine