Characterization of a FourU RNA Thermometer in the 5′ Untranslated Region of Autolysin Gene blyA in the Bacillus subtilis 168 Prophage SPβ

Abstract

RNA thermometers are noncoding RNA structures located in the 5′ untranslated regions (UTRs) of genes that regulate gene expression through temperature-dependent conformational changes. The fourU class of RNA thermometers contains a specific motif in which four consecutive uracil nucleotides are predicted to base pair with the Shine-Dalgarno (SD) sequence in a stem. We employed a bioinformatic search to discover a fourU RNA thermometer in the 5′-UTR of the blyA gene of the Bacillus subtilis phage SPβc2, a bacteriophage that infects B. subtilis 168. blyA encodes an autolysin enzyme, N-acetylmuramoyl-l-alanine amidase, which is involved in the lytic life cycle of the SPβ prophage. We have biochemically validated the predicted RNA thermometer in the 5′-UTR of the blyA gene. Our study suggests that RNA thermometers may play an underappreciated yet critical role in the lytic life cycle of bacteriophages.

Robo-Therm, a pipeline to RNA Thermometer discovery and validation

“…We used RNArobo because of its flexibility in sequence input, completely defined by the user, allowing researchers to fully handcraft and feature components of an RNA that are believed to be crucial to its function. RNArobo, or its predecessor RNAbob, has been used to discover ribozymes in various genomes, and we recently used it to discover the blyA fourU RNA thermometer that occurs in the genomes of the SPβ prophage and its host, B. subtilis 168 ( Webb et al 2009 ; Webb and Lupták 2018 ; Tong et al 2023 ). RNArobo is uniquely amenable to the discovery of RNAs that are more conserved in secondary structure than sequence, making it an efficient tool for RNA thermometer discovery.…”

“…1 ). Through this pipeline, we have previously identified and validated a fourU RNA thermometer ( Tong et al 2023 ), and here we report two novel fourU RNA thermometers that occur across different prokaryotic genomes. The first occurs upstream of a tetracycline resistance gene ( tetR ) in the intestinal bacteria Escherichia coli and Shigella flexneri .…”

“…RNA thermometers are highly structured RNA elements existing in the 5′-untranslated regions (UTRs) of mRNAs ( Altuvia et al 1989 ; Loh et al 2018 ; Sharma et al 2022 ). In most characterized examples, RNA thermometers increase gene expression of a downstream gene in response to higher temperatures ( Kortmann and Narberhaus 2012 ; Tong et al 2023 ). Most RNA thermometers function by folding into a stable secondary structure at physiologically low temperatures (<30°C) to sequester the Shine–Dalgarno (SD) sequence in a stem, preventing the ribosome from accessing the SD.…”

“…FourU thermometers contain one to four hairpins, with the terminal hairpin having at least four consecutive uracil nucleotides base-pairing with the SD sequence ( Waldminghaus et al 2007b ; Noll et al 2021 ). The fourU motif typically contains at least two noncanonical G•U wobble base pairs ( Waldminghaus et al 2007b ; Noll et al 2021 ; Tong et al 2023 ). These noncanonical base pairs contribute to the destabilization of the terminal hairpin upon heat stress; this causes the hairpin to melt and denature in a zipper-like mechanism ( Kortmann and Narberhaus 2012 ).…”

“…Bioinformatics has been used to identify novel RNA thermometers across multiple genomes ( Narberhaus et al 2006 ; Waldminghaus et al 2007a ; Chursov et al 2013 ; Churkin et al 2014 ; Tong et al 2023 ). These approaches emphasize predicted secondary structure searching due to higher conservation of secondary structure rather than sequence identity among RNA thermometers.…”