Abstract 1810 Optimization of RNA display for genetic detection of bacterial RNA-protein interactions

Research on bacterial virulence has revealed the importance of small RNAs (sRNAs), which assist in bacterial gene regulation through interactions with 5' untranslated regions (UTRs) of messenger RNAs (mRNAs). These interactions, often facilitated by RNA-chaperone proteins, are involved in bacterial growth, stress responses, and virulence, making it important to understand their molecular mechanisms. One promising approach to dissecting the mechanisms of bacterial RNA-protein interactions is a bacterial three-hybrid (B3H) assay developed by our laboratory. This system detects these interactions inside living E. coli cells by connecting the strength of RNA-protein interaction to the expression of a reporter gene. Previous studies demonstrate the B3H assay's potential to detect interactions between many sRNAs and 3'UTRs with the RNA-chaperone proteins Hfq and ProQ. Still, new constructs are needed to examine interactions with certain classes of RNAs such as 5'UTRs and RNAs with a tendency to misfold. To increase control over how RNAs are expressed and presented within the cell, we are designing novel constructs to promote the correct folding of RNA and provide control over the display of 5'UTRs. These constructs should greatly improve the scope of RNA-protein interactions that can be studied using the B3H assay. We gratefully acknowledge funding support from the Beckman Foundation, NIH (R15GM135878), the Camille and Henry Dreyfus Foundation, and Mount Holyoke College.