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.

Figure 1

Discovery of an RNA thermometer upstream of blyA. Genome loci of (A) B. subtilis SPβ bacteriophage and (B) B. subtilis 168 showing the RNA thermometer upstream of the gene blyA that encodes an N-acetylmuramoyl-l-alanine amidase. (C) Secondary structure prediction of the 5′-UTR of blyA. The fourU U41 and U42 residues and start codon are underlined, and the Shine-Dalgarno (SD) sequence is boxed. (D) Heat induction factor of bgaB fusions with the blyA 5′-UTR. Expression at 25, 37, and 42 °C was compared to a positive control, the agsA fourU RNA thermometer, and a negative control, DNA gyrase (gyrA) (mean ± standard deviation; n = 3 biological replicates). (E) Relative transcript levels of the 5′-UTR of blyA at 25 and 42 °C measured by qRT-PCR. The transcript levels of the 5′-UTR of blyA were normalized to reference gene gyrA (mean ± standard deviation; n = 3 biological replicates, each with 3 technical replicates).

Figure 2

Characterization of the blyA 5′-UTR. (A) Secondary structure prediction of the 5′-UTR of blyA, showing mutated nucleotides. The start codon is underlined, and the Shine-Dalgarno (SD) sequence is boxed. (B) Heat induction factor of bgaB fusions with mutations of the blyA 5′-UTR. Expression at 25 and 42 °C of mutants was compared to that of negative control DNA gyrase (gyrA) (mean ± standard deviation; n = 3 biological replicates). (C) Circular dichroism thermal analysis of the wild-type 5′-UTR of blyA (orange) and mutant variant UU4142CC (green) recorded at 290 nm.

Figure 3

Structural probing analysis of the 5′-UTR of blyA. (A) SHAPE reactivity plots for the 5′-UTR of blyA at 25 °C (blue) and 42 °C (red) (mean ± standard deviation; n = 3 technical replicates). U41 and U42 are underlined. (B) Secondary structure predictions of the 5′-UTR of blyA with SHAPE reactivity values at 25 and 42 °C. Nucleotides are color-coded on the basis of the intensity of SHAPE reactivity. U41 and U42 are underlined.

Figure 4

Fold induction of the 5′-UTR of mini-blyA. (A) Secondary structure prediction of the truncated 5′-UTR of blyA (mini-blyA), consisting of only the P2 hairpin. The start codon is underlined, and the Shine-Dalgarno (SD) sequence is boxed. (B) Heat induction factor comparison of bgaB fusions with the blyA and mini-blyA 5′-UTR. Expression was measured at 25, 37, and 42 °C (mean ± standard deviation; n = 3 biological replicates).