Abstract 1881 Discovery of RNA thermometers through ROBOTHERM

The Abdelsayed research group has developed a pipeline, ROBOTHERM, that utilizes bioinformatic-based structure searches to discover novel RNA thermometers across various genomes and biochemically validate their function. Most RNA thermometers regulate gene expression by increasing the expression of a downstream gene when temperature increases. Typically, temperature-induced changes in RNA secondary structure modify the availability of a sequence necessary for gene expression, such as a ribosome binding site. Examples of well-characterized RNA thermometer motifs discovered in bacteria include the fourU and ROSE-like motifs. The homology amongst these well-characterized RNA thermometer motifs was exploited as a scaffold to search for RNA thermometers with similar motifs and functions. The current bioinformatics-based approach utilizes RNARobo, an RNA motif descriptor program, to search through annotated genomes for RNA thermometers. Our new pipeline, ROBOTHERM, successfully identified many candidate RNA thermometers based on the secondary structure of previously identified RNA thermometers. Temperature-dependent expression was determined using a well-established heat-stable β-galactosidase (bgaB) reporter system to validate RNA thermometers. Through this approach, we have successfully identified and validated four new RNA thermometers in a variety of bacteria. These thermometers are found in the 5' UTR genes, including a σ factor (σ70), a DNA repair protein (RadC), an RNA helicase (DEAD/DEAH box), and a tetracycline resistance protein (TetR). The development and successful application of the ROBOTHERM pipeline underscore its efficacy in not only identifying previously unannotated RNA thermometers but also in validating their functionality across diverse bacterial genomes, contributing valuable insights into mechanisms governing heat stress through temperature-dependent gene expression. Funding provided by California Lutheran University and the McNair Scholars fund.