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Abstract

The gut microbiome is a complex habitat with many bacterial species, each playing crucial roles in regulating various physiological processes in the body. As the use of probiotics to combat human disease continues to increase, it is important to understand the mechanisms by which probiotic bacteria regulate their interactions with other bacteria and their host. Our exploration of the physiological functions of probiotic bacteria hopes to elucidate the role of small regulatory RNA (sRNA) in regulating gene expression within the microbiome. The goal of this project was to characterize the structure and function of the sRNA, UspS, which is found in probiotic, lactic acid bacteria. In Lactobacillus, UspS is closely associated with a downstream universal stress protein and contains an orphaned Lacto-usp RNA motif of unknown function. Computational methods have been used to study the UspS sRNA sequences from two Lactobacillus species in order to predict the secondary structures, generate 3D models, and search for potential mRNA interactions. Comparative sequence alignments and covariance analysis within the secondary structures predict a pseudoknot structure. The UspS sequence was isolated from two Lactobacillus species and sRNAs were synthesized by in vitro transcription with a T7 RNA polymerase. In preliminary studies, differential scanning fluorimetry of the UspS sRNA was able to confirm the presence of stable secondary structures. Future work will be focused on the structure of the pseudoknot region of UspS and its role in regulating the expression of the downstream universal stress protein.

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