title: High-resolution specificity from DNA sequencing highlights alternative modes of Lac repressor binding.
abstract: Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressor-operator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection.
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63108-8510.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zuo
authors.affiliation: Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri 63108-8510 stormo@wustl.edu.
authors.forename: Gary D
authors.identifier: http://orcid.org/0000-0001-6896-1850
authors.initials: GD
authors.lastname: Stormo
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015723:Gene Library; D056968:Lac Repressors / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017422:Sequence Analysis, DNA; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D056968:Lac Repressors
keywords: lac repressor; protein–DNA interaction; specificity by sequencing