While RNA has been heavily researched in the field of science, RNA thermometers are historically understudied. An RNA thermometer is a molecule of non-coding RNA that regulates gene expression. Bacteria are able to adapt to a wide range of both environments and temperatures. RNA thermometers (RNATs) control gene expression at the translational level. RNA thermometers enable the bacteria to respond quickly to heat and cold shock conditions through changes in RNA structure. The specific RNA thermometer that my research will be focused on is a hybrid thermometer known as Mango Froyo. A synthetic hybrid was made with a known RNA thermometer, called blyA, combined with an aptamer called RNA Mango. In cold temperatures, the ribosome binding site is unavailable because of the thermometer’s rigid structure, but with added heat, the thermometer opens up and allows the ribosome to bind. The conical ligand for the mango aptamer is TO-1. The TO-1 dye only fluoresces when bound to the Mango aptamer. Our specific aim is to develop and test a synthetic platform of a thermometer that provides fluorescent feedback in response to a change in expression. The goal is to characterize Mango Froyo by using various methods including beta-galactosidase assays. The main result that was found during our research was that we were able to validate the thermometer function. Future researchers can use the synthetic tool, an RNA thermometer, to regulate gene expression and to control and track RNA expression upon heat induction.

RNA thermometers are a temperature-sensitive non-coding RNA molecule that regulate gene expression through either heat shock or cold shock response. RNA thermometers are temperature sensitive, at cold temperatures the ribosome binding (RBS) site is trapped and translation of mRNA is turned off. Upon heat induction, the RBS is available and translation is on. The Rose DEAD/DEAH box helicase gene has a potential RNA thermometer in the 5’ untranslated region (5’-UTR). The ROSE DEAD/DEAH box helicase contributes to innate immune signaling and remodels misfolded RNA structures. The purpose of this study is to test the predicted RNA thermometer in front of the Rose DEAD/DEAH box helicase gene to see if more protein can be produced upon heat induction, changing the gene expression. To observe these effects, we designed a specific thermometer sequence, performed PCR tests, Hifi assembly cloning, and performed Beta-galactosidase assays. Beta-galactosidase assays suggest that successful results will show increased enzymatic activity, proving that there is more beta-galactosidase present due to temperature. The more beta-galactosidase present will prove that the RNA thermometer is working with heat change. Although the proposed ROSE helicase was successfully cloned, the sequence used was not the full sequence necessary to correctly test the 5’UTR . Future experiments involving beta-galactosidase assays will be necessary to determine preliminary findings. Findings reflected the control group and future directions will include obtaining a new sequence and performing triplicates.

RNA is heavily researched in the field of science, but RNA thermometers are historically understudied. An RNA thermometer is a molecule of non-coding RNA that regulates gene expression. Bacteria are able to adapt to many environments and temperatures. RNA thermometers control gene expression at the translational level. RNA thermometers enable the bacteria to respond quickly to heat/cold shock conditions through changes in RNA structure. The specific RNA thermometer that my research will focus on is a hybrid thermometer known as Mango Froyo. A synthetic hybrid was made with an RNA thermometer, BlyA, combined with an aptamer called RNA Mango. In cold temperatures, the ribosome binding site is unavailable because of the rigid structure; added heat allows the thermometer to open up and allows the ribosome to bind. The conical ligand for the mango aptamer is TO-1. The TO-1 dye only fluoresces when bound to the Mango aptamer. My specific aim is to test the fluorescence of the aptamer and test a synthetic platform of the thermometer that provides fluorescent feedback. My goal is to characterize Mango Froyo by using various methods, including PCR reactions, fluorescence assays, and beta-galactosidase assays. The main result that was found during my research was that we were able to validate the thermometer function. To study the synthetic fluorescent RNA thermometer Mango Froyo would bring great significance for research and clinical implications. Future researchers can use the synthetic tool, RNA thermometers, to regulate gene expression and to control and track RNA expression upon heat induction.

RNA Thermometers are heat-regulated novel RNA structures that play a vital role in the regulation of protein synthesis. RNAs are generally single stranded molecules, but thermometers are hairpin structures that base-pair on themselves, constricting ribosome binding through sequestering the ribosome binding site (RBS). When introduced to higher temperatures, the hairpin containing the RBS denatures, allowing binding to a ribosome to complete translation. Using bioinformatics programs, RNArobo and BLAST, an RNA thermometer structure, similar to an established thermometer, was discovered in front of the gene encoding for RadC protein in Staphylococcus epidermidis RP62A. The hypothesized RNA sequence was inserted into a DNA plasmid that was, first, amplified and linearized using polymerase chain reaction, PCR. Then, HIFI DNA Assembly kit was utilized to clone the plasmid that had the insert sequence in it. The cloned plasmid was inserted into a cultured bacteria colony and beta-galactosidase assays were performed to test if the hypothesized sequence is a functional RNA thermometer. Assay results of fluorescent absorbances showed a 2.78 fold induction from 25°C to 37°C and 4.58 fold induction from 37°C to 42°C. Thus, gene expression of beta-galactosidase was induced as temperature increased, leading to the conclusion that the RadC protein is heat regulated by a RNA thermometer.