Optimization of RNA display for genetic detection of bacterial RNA-protein interactions
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Abstract
Research on bacterial virulence has revealed the importance of small RNAs (sRNAs), which assist in bacterial gene regulation through interactions with 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. Previous work has shown that an exogenous terminator provided to 5'UTR-containing bait constructs causes a detrimental background signal in the B3H. To increase control over how RNAs are expressed and presented within the cell, we are designing novel constructs to provide control over the display of 5'UTRs and prevent background signal. I have focused on affording post-transcriptional control over the RNA bait construct by introducing modifications that would remove the terminator sequence hypothesized to be responsible for background interaction. In this work, I demonstrated that a modified bait RNA construct that contains a self-cleaving ribozyme is capable of decreasing background signal in the B3H assay. Modification of the RNA bait construct to contain a ribozyme enables us to examine what ribozyme classes are capable of cleaving in vivo, as well as mRNA degradation after post-transcriptional processing. While results indicate the ribozyme-modified RNA bait construct is effective in reducing background signal interaction when no 5’UTR is present in the construct, for the application of detecting a broader range of RNA-protein interactions, it was imperative to determine if the presence of a ribozyme disrupted established 5’UTR-protein interactions. My results suggest that a ribozyme in the context of the RNA bait construct cleaves in vivo and does not disrupt 5’UTR-protein interactions. These constructs should greatly improve the scope of RNA-protein interactions that can be studied using the B3H assay.
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RNA, genetics, gene regulation