RNA thermometers are molecules comprised of non-coding RNA that are able to regulate gene expression at the translational level through changes in temperature. This is because bacteria require specific temperatures to survive in, so they are able to respond to heat and cold shock conditions. Along with my lab partners, we will be focusing on the synthetic thermometer Mango Froyo, which is a hybrid of a known thermometer, blyA, and an RNA aptamer, Mango aptamer. The Mango aptamer has the ability to bind to TO-1 biotin, which allows it to fluoresce when bound. Knowing this background, our goal in this project is to determine if temperature affects the structure and function of the hybrid Mango thermometer. We also aim to determine if the thermometer, in combination with TO-1 biotin, is able to make RNA fluoresce, and how that would affect binding and the structure of Mango Froyo. Through various biological and biochemical methods, such as DNA amplification, serial dilutions, and beta-galactosidase assays, we were able to measure the amount of reactivity of enzymes. These results have allowed us to verify the function of Mango Froyo as an RNA thermometer and aptamer fluorescence function. Future projects for our lab group include attempting to transcribe RNA in the presence of TO-1 and perform further experiments on fluorescence. This is a very understudied field in biology and biochemistry but could have significant clinical implications, RNA thermometers could help regulate gene expression and could allow RNA to be tracked in real time.

This study investigated the activity of a potential fourU RNA thermometer, upstream of the RNA polymerase Sigma 70 factor, under three distinct temperature conditions (25°, 37°C, and 42°C). This research aims to investigate if the 5’ untranslated region (5’-UTR) of sigma 70 regulates the expression of sigma 70 in a temperature-dependent manner. RNA thermometers are genetic control systems that utilize RNA to detect alterations in temperature. When exposed to lower temperatures, the messenger RNA (mRNA) assumes a specific structure that conceals the ribosome binding site, known as the Shine-Dalgarno (SD) sequence, located within the 5' untranslated region (5'-UTR). This structural change effectively obstructs ribosome binding and the subsequent process of translation. The σ70 family of sigma factors is a crucial part of the RNA polymerase, responsible for guiding the bacterial to its specific promoter regions, and initiate transcription. We characterized the impact of temperature increase on the RNA thermometer's functionality by cloning into bacteria cells and testing the expression with a reporter plasmid containing beta-galactosidase. These investigations were performed to explore the implications of RNA thermometers in genomics discovery and elucidate bacterial adaptation mechanisms in response to environmental changes. Initial results demonstrate that the 5’UTR of sigma 70 is an RNA thermometer.

Ribonucleic acid thermometers are RNA sequences that are temperature sensitive and help regulate gene expression. Typically, RNA thermometers undergo a conformational change upon heat stress, resulting in an upregulation of a gene downstream of the thermometer sequence. The TetR-family of transcriptional regulators (TFTRs) are a large family of signal transduction proteins that are implicated in the regulation of many processes, including cell division and the stress response in cells. In this study, we used a FourU RNA thermometer to analyze the effect it has on gene expression with the TetR family transcriptional regulator C-terminal domain-containing protein. We used PCR, HIFI assembly, Beta-galactosidase assay, and cloning to undergo this experiment. Preliminary results indicate and increase in expression in response to heat induction. This is presumably due to the zipper-like melting of a motif containing the ribosome binding site (RBS). Overall, this helps us with further development of antibiotic resistance through gene regulation.