Abstract 1336 Characterization of a ROSE-like RNA Thermometer Discovered Upstream of an ABC Transporter

RNA Thermometers (RNATs) are functional non-coding regions of RNA that regulate the expression of genes downstream in response to temperature. One particular class of RNATs, Repression Of heat Shock gene Expression (ROSE)-like thermometers, contains a conserved nucleotide motif, U(U/C)GCU, that base pairs with the Shine-Dalgarno (SD) site at low temperatures. This base pairing blocks the ribosome from binding to the SD and hinders its ability to translate the mRNA into protein, unless an increase in temperature is introduced. We recently discovered a ROSE-like RNAT associated with an ABC Transporter gene (oppF) in the bacterium Providencia stuartii. We aimed to characterize the secondary structure of that particular RNAT using Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE), as well as to determine the levels of transcriptional control using Quantitative Reverse Transcription PCR (qRT-PCR). Structure probing of the RNAT using SHAPE established a change in the reactivity profile from 25 to 42°C at a single-nucleotide resolution, suggesting that the RNAT undergoes a conformational change between the two temperatures. qRT-PCR experiments demonstrated a minimal 1.4-fold increase in RNAT transcript levels from cells incubated at 25°C in comparison to 37°C. However, no change was observed in transcript levels from 37 to 42°C, indicating that gene regulation is achieved post-transcriptionally. Our studies support the RNAT's claim as a post-transcriptional thermoregulator of gene expression, and adds new layers to our understanding of the temperature regulation mechanism of RNA thermometers that regulate ABC transporter genes. Financial support was provided by California Lutheran University (CLU) start-up funding (M.M.A.), the intramural program of the National Heart, Lung and Blood Institute, NIH (L.F.M.P.), the John Stauffer Charitable TrustFoundation - CLU (A.Y.T.) and the Swenson Science Summer Research Fellowship - CLU (E.L.T.).