Robo-Therm, a pipeline to RNA thermometer discovery and validation.

RNA thermometers are highly structured noncoding RNAs located in the 5'-untranslated regions (UTRs) of genes that regulate expression by undergoing conformational changes in response to temperature. The discovery of RNA thermometers through bioinformatics is difficult because there is little sequence conservation among their structural elements. Thus, the abundance of these thermosensitive regulatory structures remains unclear. Herein, to advance the discovery and validation of RNA thermometers, we developed Robo-Therm, a pipeline that combines an adaptive and user-friendly in silico motif search with a well-established reporter system. Through our application of Robo-Therm, we discovered two novel RNA thermometers in bacterial and bacteriophage genomes found in the human gut. One of these thermometers is present in the 5'-UTR of a gene that codes for σ 70 RNA polymerase subunit in the bacteria Mediterraneibacter gnavus and Bacteroides pectinophilus, and in the bacteriophage Caudoviricetes, which infects B. pectinophilus The other thermometer is in the 5'-UTR of a tetracycline resistance gene (tetR) in the intestinal bacteria Escherichia coli and Shigella flexneri Our Robo-Therm pipeline can be applied to discover multiple RNA thermometers across various genomes.

Characterization of a FourU RNA Thermometer in the 5' Untranslated Region of Autolysin Gene blyA in the Bacillus subtilis 168 Prophage SPß.

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 SPßc2, 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 SPß 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.