Vibrio parahaemolyticus has two sets of type III secretion systems that are major pathogenic factors: T3SS1 (cytotoxicity) and T3SS2 (enterotoxicity). V. parahaemolyticus mainly colonizes the distal small intestine after oral infection and may be exposed to carbon-limiting stress due to the lack of readily available carbohydrates in this environment. Catabolite activator protein (CAP), a transcription factor involved in carbon-limiting metabolism in many Gram-negative bacteria, is well known to be involved in the regulation of the expression of many virulence factors. In this study, we determined the effects of CAP on the expression of T3SSs in this bacterium. Based on a lactate dehydrogenase-based cytotoxicity assay, CAP was found to have a greater contribution to the expression of T3SS2-dependent cytotoxicity than to that of T3SS1. Reverse transcription quantitative PCR revealed decreased expression of many T3SS2-related genes, including vpa1348, in the cap gene deletion mutant compared to the parent strain. CAP was demonstrated to bind near the T-rich elements within the vpa1348 promoter region in an electrophoretic mobility shift assay and DNase I footprinting. CAP also enhanced the expression of vpa1348 in a β-galactosidase reporter assay. Collectively, these results suggest that CAP is involved in T3SS2-mediated virulence by regulating the expression of vpa1348 in V. parahaemolyticus.

Nanoparticle carriers enable the multivalent delivery of nucleic acids to cells and protect them from degradation. In this chapter, we present a comprehensive overview of four methodologies: electrophoretic mobility shift assay (EMSA), alamarBlue/CFDA-AM cell viability dyes, fluorescence microscopy, and antiviral assays, which collectively are tools to explore interactions between nucleic acids and nanoparticles, and their biological efficacy. These assays provide insights into binding potential, cytotoxicity, and antiviral efficacy of nucleic acid-based nanoparticle treatments furthering the development of effective antiviral therapeutics.

Protein-nucleic acid interactions drive some of the most important physiological events in cells. Here, we present a protocol for detecting protein-DNA or protein-RNA interactions in vitro. We describe steps for labeling nucleic acid species and electrophoretic mobility shift assays (EMSAs). This protocol can be used to confirm suspected in vivo interactions using recombinantly expressed/purified proteins of interest and a nucleic acid substrate. It can further be used to investigate mutations that can disrupt interaction or compensatory mutations that restore it. For complete details on the use and execution of this protocol, please refer to Mansouri-Noori et al.1.

Plant long non-coding RNAs (lncRNAs) have been implicated in many biological processes, including responses to abiotic stresses, yet their detailed functions and especially their modes of action are still underexplored. lncRNAs often interact with proteins to participate in multiple levels of gene regulation. Therefore, identifying the RNA-binding proteins and validating their interaction with lncRNAs will be instrumental in revealing the functions of lncRNAs. Here, we describe two major methods to determine the interaction between lncRNA and proteins in vitro, RNA pull-down, and RNA EMSA.

DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

EWSR1 (Ewing Sarcoma Related protein 1) is an RNA binding protein that is ubiquitously expressed across cell lines and involved in multiple parts of RNA processing, such as transcription, splicing, and mRNA transport. EWSR1 has also been implicated in cellular mechanisms to control formation of R-loops, a three-stranded nucleic acid structure consisting of a DNA:RNA hybrid and a displaced single-stranded DNA strand. Unscheduled R-loops result in genomic and transcription stress. Loss of function of EWSR1 functions commonly found in Ewing Sarcoma correlates with high abundance of R-loops. In this study, we investigated the mechanism for EWSR1 to recognize an R-loop structure specifically. Using electrophoretic mobility shift assays (EMSA), we detected the high affinity binding of EWSR1 to substrates representing components found in R-loops. EWSR1 specificity could be isolated to the DNA fork region, which transitions between double- and single-stranded DNA. Our data suggests that the Zinc-finger domain (ZnF) with flanking arginine and glycine rich (RGG) domains provide high affinity binding, while the RNA recognition motif (RRM) with its RGG domains offer improved specificity. This model offers a rational for EWSR1 specificity to encompass a wide range in contexts due to the DNA forks always found with R-loops.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Regulatory RNAs in bacteria are known to act by base pairing with other RNAs. Interactions between two partner RNAs can be investigated by electrophoretic mobility shift assays. The regions predicted to be engaged in base pairing are analyzed by introducing mutations in one RNA that prevent RNA-RNA complex formation. Next, base pairing is restored by introducing complementary mutations in its partner RNA. Here, we describe the mutational strategy and experimental methods used to validate specific base pairing between two RNA species.

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.