Genetic assays to define and characterize protein–protein interactions involved in gene regulation

Transcription can be regulated during initiation, elongation, and termination by an enormous variety of regulatory factors. A critical step in obtaining a mechanistic understanding of regulatory factor function is the determination of whether the regulatory factor exerts its effect through direct contact with the transcription machinery. Here I describe the application of a transcription activation-based bacterial two-hybrid assay that is useful for the identification and genetic dissection of protein–protein interactions involved in gene regulation. I provide examples of how this two-hybrid system can be adapted for the study of “global” regulatory factors, sequence-specific DNA-binding proteins, and interactions that occur between two subunits of RNA polymerase (RNAP). These assays facilitate the isolation and characterization of informative amino acid substitutions within both regulatory factors and RNAP. Furthermore, these assays often enable the study of substitutions in essential domains of RNAP that would be lethal in their natural context.