Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription.

Affiliation

Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University; Graduate Program in Physiology and Integrative Biology, School of Graduate Studies, Rutgers University; [Email]

Abstract

A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ66 and σ28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

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