Optimizing a genetic reporter for RNA-protein interactions: in vivo titration of a promoter binding protein



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Gene regulation is an essential process in all domains of life that governs which genes are expressed at a given time to control cell specialization and adaptation to environmental changes. In bacteria, non-coding small RNAs (sRNAs) regulate gene expression in response to a specific stress signal and are critical in pathways bacteria use to resist antibiotics, form biofilms, and infect other cells. sRNAs regulate gene expression, including mRNA translation and stability, by base pairing to target mRNAs through interactions with proteins, such as the bacterial RNA chaperone Hfq. However, not much is understood about the mechanism of interaction between sRNAs and proteins and new methods to study RNA-protein interactions have been developed, such as protein immunoprecipitation, to isolate proteins that interact with small RNAs. However, these approaches are limited in that purification of the protein and RNA transcript outside of the natural cellular environment may not accurately reflect in vivo interactions. An in vivo bacterial three-hybrid (B3H) assay was developed to study sRNAs and their interactions with chaperone proteins such as Hfq. The B3H assay detects the variable RNA-protein interaction by coupling the interaction to reporter gene expression, in this case the expression of β-galactosidase. When the RNA and protein of interest interact, RNA polymerase binds to the test promoter and initiates transcription of the reporter gene, lacZ. The B3H assay successfully detects an interaction between Hfq and several sRNAs. However, there are a number of additional known sRNA-Hfq interactions the assay does not reliably detect. My project focuses on modulating expression levels of the DNA-RNA adapter protein in the assay to increase the signal detected by the B3H assay. To alter protein expression, I designed several promoters of varying strength and demonstrated that the B3H signal can be modulated based on the strength of the promoter driving expression of the DNA-RNA adapter protein. I then conducted a forward genetic screen and identified a set of promoters that further increased the signal. With the combined approaches of site-directed and random mutagenesis, we show that a weak promoter driving expression of the protein component increases the signal detected by the B3H assay for several Hfq-sRNA interactions. With an optimized B3H assay, more could be learned about the regulatory roles sRNAs and protein chaperones play in biology.



Biochemistry, RNA, RNA-protein interactions, Bacterial three-hybrid assay