title: Understanding DNA-binding specificity by bacteria hybrid selection.
abstract: Understanding how sequence-specific protein-DNA interactions direct cellular function is of great interest to the research community. High-throughput methods have been developed to determine DNA-binding specificities; one such technique, the bacterial one-hybrid (B1H) system, confers advantages including ease of use, sensitivity and throughput. In this review, we describe the evolution of the B1H system as a tool capable of screening large DNA libraries to investigate protein-DNA interactions of interest. We discuss how DNA-binding specificities produced by the B1H system have been used to predict regulatory targets. Additionally, we examine how this approach has been applied to characterize two common DNA-binding domain families-homeodomains and Cys2His2 zinc fingers-both in organism-wide studies and with synthetic approaches. In the case of the former, the B1H system has produced large catalogs of protein specificity and nuanced information about previously recovered DNA targets, thereby improving our understanding of these proteins' functions in vivo and increasing our capacity to predict similar interactions in other species. In the latter, synthetic screens of the same DNA-binding domains have further refined our models of specificity, through analyzing comprehensive libraries to uncover all proteins able to bind a complete set of targets, and, for instance, exploring how context-in the form of domain position within the parent protein-may affect specificity. Finally, we recognize the limitations of the B1H system and discuss its potential for use in the production of designer proteins and in studies of protein-protein interactions.
authors.forename: Denise J
authors.initials: DJ
authors.lastname: Xu
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Noyes
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D014157:Transcription Factors; D004247:DNA
keywords: DNA-binding domains; DNA-binding specificity; bacterial hybrid assay; protein engineering; protein selection systems; transcription factor