Detection of Transcription Factor Partners with a Yeast One Hybrid Screen

Abstract

Transcription factors usually engage in multiple regulatory interactions with other proteins in their normal promoter or enhancer context. Conventional yeast two hybrid screens, in which a putative protein interaction domain is fused to a heterologous DNA binding domain like that of LexA or Gal4 (1–3) have been used extensively to detect protein/protein interactions. Even though the technique has also identified partners of transcription factors (for example [3–5]), it has particular limitations in this application. This is because many transcription factors undergo conformational changes upon DNA binding and frequently engage in interactions with other factors through surfaces on their DNA-binding domains. When target factors are fused to the heterologous Gal4 or LexA DNA binding domains and exposed in their non-DNA-bound form, relevant interaction epitopes may be shielded or inappropriately folded. By contrast, in a situation where the transcription factor is bound to its authentic binding site on the DNA via its own DNA binding domain, its conformation should closely resemble that found in a normal promoter context and thus detect functionally relevant interactions with other factors. This concept has been instrumental in identifying coactivators of transcription factors that are transcriptionally inert in yeast (6–8). The majority of transcription factors, however, have transactivation potential in yeast. Therefore the author has designed a generally applicable yeast one hybrid screen that uses a transcriptionally disabled target protein to take advantage of the normal conformation of a DNA bound transcription factor (see Fig. 1).