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Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system

Nature Protocols volume 1, pages 30–45 (2006)Cite this article

Abstract

Bacterial-based interaction trap systems provide a powerful method to identify interacting macromolecules. When carried out in the context of a genetic selection, interacting pairs can be rapidly isolated from large combinatorial libraries. This technology has been adapted to allow the identification of DNA-binding sequences for a transcription factor (TF) from a large randomized library. This procedure uses a library of randomized binding sites upstream of a cocistronic HIS3-URA3 reporter cassette. The URA3 reporter allows self-activating sequences to be removed from the library through counter-selection. The HIS3 reporter allows sequences that are recognized by a TF to be isolated from the library, where transcriptional activation is mediated by fusion of the TF to the α-subunit of RNA polymerase. This technology can be used to characterize monomeric, homodimeric and heterodimeric DNA-binding domains and, once a suitable library is constructed, binding sites can be identified in approximately 10 d. The bacterial one-hybrid system allows larger libraries to be searched than the corresponding yeast one-hybrid system and, unlike SELEX, it does not require purification of the TF(s). The complexity of the binding site libraries that can be searched using the bacterial system is, however, more limited than SELEX, and some eukaryotic factors may not express or fold efficiently in the bacterial system.

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Figure 1: Overview of the bacterial one-hybrid selection system.
Figure 2: A flow chart with approximate timelines for (a) the binding-site library construction and purification, (b) the selection procedure to isolate TF binding sites from a purified binding-site library and (c) an additional counter-selection to eliminate self-activating sequences.
Figure 3: Plasmid maps and detailed information on the multiple cloning sites (MCS) present in the plasmids for the bacterial one-hybrid system.
Figure 4: Example of the process used to identify overrepresented sequence motifs present in insert sequences isolated from a binding-site selection.

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Acknowledgements

We would like to thank J. McNulty and M. Noyes for critiquing this manuscript. This work was supported by a National Institutes of Health grant 1R01GM068110.

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Authors and Affiliations

  1. Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street, Worcester, 01605, Massachusetts, USA

    Xiangdong Meng & Scot A Wolfe

  2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, 01605, Massachusetts, USA

    Scot A Wolfe

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Correspondence to Scot A Wolfe.

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The authors have a pending patent application on related subject matter (USPTO).

Supplementary information

Supplementary Figure 1

Self-activating sequences isolated from the binding site selections. (PDF 3712 kb)

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Meng, X., Wolfe, S. Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system. Nat Protoc 1, 30–45 (2006). https://doi.org/10.1038/nprot.2006.6

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