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

Summary

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

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.

Introduction

Transcription, which produces RNA molecules using DNA as template, is the very first step of gene expression. Bacterial RNA synthesis begins following the binding of the RNA polymerase (RNAP) holoenzyme to a target promoter^1,2. The RNAP holoenzyme (RNAPholo) is comprised of a multi-subunit catalytic core (RNAPcore) and a σ factor, which is required for recognizing the promoter sequence. Transcription activators and repressors, collectively termed TFs, regulate the gene expression through the binding components of the RNAPcore, σ factors, and/or DNA. Depending on the organism, a significant portion of....

Protocol

1. Preparation of proteins

1. Use a dialysis bag (with an appropriate cut-off size) to dialyze each protein to be used for BLI assays (including both the His-tagged ligand and the analyte) against 1,000 volumes of the BLI buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA, 10 mM MgCl₂, 0.1 mM DTT, pH 8.0, pre-chilled to 4 ˚C) at 4 ˚C for 4 h.
   NOTE: BLI assays require the ligand to be present at concentrations that saturate the binding sites on the biosensor and the analyte to be highly purified so that the molar concentrations of the analyte that react with the ligand is known. Methodologies for the expression and purification of His....

Results

Through BLI assays, we previously established that binding of GrgA to σ²⁸ is dependent on a 28 amino acid middle region (residues 138-165) of GrgA¹⁵. Accordingly, compared with N-terminally His-tagged full length GrgA (NH-GrgA), a GrgA deletion construct lacking this region (NH-GrgAΔ138-165) had a decreased association rate and an increased dissociation rate, leading to a 3 million-fold loss of overall affinity (Table 1). Here, we demonstrate that this middle region dir.......

Discussion

Protein-protein interactions are crucial for the regulation of transcription and other biological processes. They are most commonly studied through pulldown assays. Although pulldown assays are relatively easy to perform, they are poorly quantitative and may fail to detect weak but biologically meaningful interactions. In comparison, by detecting real-time association and dissociation between a ligand and an analyte, BLI provides association and dissociation rate constants, as well as, overall affinity.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BLItz machine	ForteBio	45-5000
Dialysis tubing cellulose membrane	MilliporeSigma	D9652
Dip and Read Ni-NTA biosensor tray	ForteBio	18-5101	Ready-to-use Ni-NTA biosensors for poly-His-tagged Proteins
Drop holder	ForteBio	45-5004
PCR tubes (0.2 mL)	Thomas Scientific	CLS6571
Microcentrifuge tubes (black)	Thermo Fisher Scientific	03-391-166
Kimwipes	Thermo Fisher Scientific	06-666A
DTT	Thermo Fisher Scientific	R0861
EDTA	MilliporeSigma	E6758
MgCl2	MilliporeSigma	M8266
NaCl	MilliporeSigma	S9888
Tris-HCl	GoldBio	T095100