Biology subjects

Berry, K. E.

Publications and source records attributed to Berry, K. E..

8 recordsLinked to original sources

bioRxiv2026

Interdependent mode of RNA recognition by the interacting KH-domain proteins KhpA and KhpB

RNA-binding proteins (RBPs) are central regulators of gene expression. In bacteria, two KH domain-containing proteins, KhpA and KhpB, are increasingly recognized as globally-acting RBPs, including in pathogens that lack well-studied RBPs, such as Hfq and ProQ. While KhpA and KhpB interact with various RNAs in diverse bacteria, their crosstalk and molecular basis of RNA recognition remain poorly understood. Here, we explored the mutual dependence between KhpA and KhpB in the food-borne pathogen Campylobacter jejuni. We demonstrate KhpA and KhpB interact in vivo, and their individual global RNA recognition is largely dependent on the presence of the partner protein. Using RNA co-immunoprecipitation, we reveal that KhpA requires a conserved KH domain motif for RNA binding, whereas KhpB relies on an atypical R3H domain. Moreover, mutating the RNA-binding domain of one protein compromises the RNA interactome of the other. Overall, we demonstrate an interdependent RNA-binding mode of KhpA and KhpB and anticipate that such KH-proteins have co-evolved to elicit joint RNA regulation in bacteria.

microbiology↗

bioRxiv2026

Genetic detection of RNA-protein interactions using a bacterial three-hybrid assay

The bacterial three-hybrid (B3H) assay is a powerful genetic tool for detecting interactions between RNA and RNA-binding proteins (RBPs) and assessing the consequences of RBP mutations. This transcription-based system connects the strength of an RNA-protein interaction to the expression of a lacZ reporter gene in Escherichia coli cells. This in vivo approach allows researchers to dissect RNA-protein interactions within a cellular environment, bypassing the need for biochemical purification of RNAs or proteins. This chapter details a three-day protocol for generating quantitative B3H data. Since a significant challenge in B3H assays is RNA misfolding, we describe a recently optimized set of B3H constructs that mitigates this issue by isolating bait RNAs as discrete folding units.

molecular biology↗

bioRxiv2026

Molecular genetic characterization of bacterial KH-domain proteins

Post-transcriptional gene regulation is a key mechanism for bacterial stress responses and virulence, and RNA-mediated regulation frequently relies on global RNA-binding proteins (RBPs). A pair of interacting KH-domain proteins (KhpA and KhpB) have recently been identified as global RBPs in several bacterial species. To better understand their molecular functions, we employed bacterial two- and three-hybrid (B2H/B3H) assays in an E. coli reporter system to analyze protein-protein and protein-RNA interactions of KhpA/B orthologs from three human pathogens: Campylobacter jejuni, Helicobacter pylori, and Clostridioides difficile. Protein-protein interactions were conserved across all species, with KhpA-KhpB heterodimers forming more robustly than either homodimer and KhpA homodimerizing more readily than KhpB. On the other hand, protein-RNA interactions were more varied across species: C. jejuni and C. difficile KhpA bound both species-specific and non-specific RNAs, but H. pylori KhpA--and KhpB orthologs from all species--showed no RNA interaction in B3H assays. Site-directed mutagenesis experiments demonstrated that residues in the GXXG motif of KhpA are critical for RNA interaction and differences in these residues account for the distinct RNA-binding behaviors of KhpA orthologs. Collectively, these findings provide a cross-species, molecular view of how KhpA and KhpB recognize one another and RNA ligands to regulate gene expression.

molecular biology↗

bioRxiv2024

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

We have previously developed a transcription-based bacterial three-hybrid (B3H) assay as a genetic approach to probe RNA-protein interactions inside of E. coli cells. This system offers a straightforward path to identify and assess the consequences of mutations in RBPs with molecular phenotypes of interest. One limiting factor in detecting RNA-protein interactions in the B3H assay is RNA misfolding arising from incorrect base-pair interactions with neighboring RNA sequences in a hybrid RNA. To support correct folding of hybrid bait RNAs, we have explored the use of a highly stable stem ("GC clamp") to isolate regions of a hybrid RNA as discrete folding units. In this work, we introduce new bait RNA constructs to 1) insulate the folding of individual components of the hybrid RNA with GC clamps and 2) express bait RNAs that do not encode their own intrinsic terminator. We find that short GC clamps (5 or 7 bp long) are more effective than a longer 13bp GC clamp in the B3H assay. These new constructs increase the number of Hfq-sRNA and -5'UTR interactions that are detectable in the B3H system and improve the signal-to-noise ratio of many of these interactions. We therefore recommend the use of constructs containing short GC clamps for the expression of future B3H bait RNAs. With these new constructs, a broader range of RNA-protein interactions are detectable in the B3H assay, expanding the utility and impact of this genetic tool as a platform to search for and interrogate mechanisms of additional RNA-protein interactions.

molecular biology↗

bioRxiv2023

Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli, though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQs FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNAs terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70 and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

molecular biology↗

bioRxiv2020

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

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 RNA-chaperone 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.

microbiology↗

bioRxiv2020

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least two base pairs preceding an 3 oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5 side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3 and cspE-3 RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3 intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.

molecular biology↗

bioRxiv2019

Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs. Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex.

biochemistry↗