Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA–DNA adapter protein

Abstract

Non-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNAchaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system and will facilitate exploration of the binding mechanisms of these interactions.

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofEscherichia coliProQ

“…1 B); the strength of an RNA–protein interaction correlates to the fold-stimulation in β-gal activity over basal levels when all components are present ( Fig. 1 C; Wang et al 2021 ; Stockert et al 2022 ).…”

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain ofEscherichia coliProQ

“…Reporter cells encode a lacZ reporter gene downstream of a test promoter on a single-copy Fʹ episome. Transformation of reporter cells with all three plasmids (pPrey, pAdapter, and pBait) leads to a boost in β-gal levels relative to basal levels indicated by three negative controls in which half of each hybrid component is left out (Fig 1B); the strength of an RNA-protein interaction correlates to the fold-stimulation in β-gal activity over basal levels when all components are present (Fig 1C; (Wang et al 2021;Stockert et al 2022)).…”

Improved constructs for bait RNA display in a bacterial three-hybrid assay

“…While there is substantial potential in the B3H assay, its current utility has been limited by only a modest success rate in detecting established RNA-protein interactions. Improvements in the expression level of adapter protein have increased signal for several Hfq-sRNA interactions with KD values out to ~100 nM (Wang et al 2021). Despite this progress, many well-characterized Hfq-sRNA interactions are undetectable in the current B3H assay.…”

“…While the original pBait constructs contained two copies of the MS2 hp (Berry and Hochschild 2018;Wang et al 2021), a single MS2 hp has been shown to be sufficient for detection of Hfq-sRNA interactions and, indeed, is critical for accurate detection of ProQ-RNA interactions (Pandey et al 2020). Due to its simplicity and broader applicability, we chose to focus our optimization of RNA display here on the 1xMS2 hp pBait construct.…”

“…This bacterial three-hybrid (B3H) assay utilizes E. coli's cellular machinery to express hybrid RNAs and proteins in vivo, avoiding the need to purify these components and allowing them to be studied in native cytoplasmic conditions. We have used the B3H system to study the interactions of both E. coli Hfq and ProQ (Berry and Hochschild 2018;Pandey et al 2020;Wang et al 2021;Stein et al 2020Stein et al , 2023. As the assay affords access to powerful tools of molecular genetics, it supports the dissection of molecular mechanisms using unbiased mutagenesis screens, as well as the potential discovery of novel RBPs.…”