Transcriptome-wide probing reveals RNA thermometers that regulate translation of glycerol permease genes in Bacillus subtilis

Abstract

RNA structure regulates bacterial gene expression by several distinct mechanisms via environmental and cellular stimuli, one of which is temperature. While some genome-wide studies have focused on heat shock treatments and the subsequent transcriptomic changes, soil bacteria are less likely to experience such rapid and extreme temperature changes. Though RNA thermometers (RNATs) have been found in 5' untranslated leader regions (5' UTRs) of heat shock and virulence-associated genes, this RNA-controlled mechanism could regulate other genes as well. Using Structure-seq2 and the chemical probe dimethyl sulfate (DMS) at four growth temperatures ranging from 23°C to 42°C, we captured a dynamic response of the Bacillus subtilis transcriptome to temperature. Our transcriptome-wide results show RNA structural changes across all four temperatures and reveal nonmonotonic reactivity trends with increasing temperature. Then, focusing on subregions likely to contain regulatory RNAs, we examined 5' UTRs to identify large, local reactivity changes. This approach led to the discovery of RNATs that control the expression of glpF (glycerol permease) and glpT (glycerol-3-phosphate permease); expression of both genes increased with increased temperature. Results with mutant RNATs indicate that both genes are controlled at the translational level. Increased import of glycerols at high temperatures could provide thermoprotection to proteins.

Keywords: RNA thermometers; RNA–protein; Structure-seq; glycerol permease.

FIGURE 1.

Schematic for DMS treatment, library quality, data analysis, and reporter assays. (A) Generation of DMS-induced reverse transcription (RT) stops in Bacillus subtilis grown at one of four different temperatures. RNA samples are then prepped for Illumina sequencing using Structure-seq2. (B) Comparison of RT stops in three DMS-treated biological replicates grown at 37°C. Pearson correlation coefficients (r) are shown. (C) Schematic representing a 5′ untranslated region (5′ UTR) with unstructured (1 and 3) and structured (2) regions. (D) Reactivity profile of the 5′ UTR from (C) plotted for each temperature. (E) Schematic of RNAT-controlled translation initiation. At low temperatures, the ribosome binding site (RBS) is sequestered, and the ribosome (purple) does not bind. At high temperatures, the RBS is available for the ribosome to bind and initiate translation of the downstream gene.

FIGURE 2.

Transcriptome-wide changes in reactivity across four growth temperatures in B. subtilis. (A) Mean structurome changes in reactivity for all transcripts at 23°C (blue), 30°C (purple), 37°C (orange), and 42°C (yellow) shown as violin plots with box plots in the center. Gray spider lines link individual transcript reactivity in each condition. Global mean values are given below the violin plot for each temperature, as well as standard deviation (sd) values. Additionally, t-test P-values are given between the compared data sets. Mean reactivity increased from 23°C to 30°C, and further increased at 37°C, then slightly decreased at 42°C. (B–D) Scatter plots showing Δ reactivity (from higher temperature to lower temperature) against Δ abundance (from higher temperature to lower temperature) for (B) 23°C–30°C, (C) 30°C–37°C, and (D) 37°C–42°C. All plots show an inverse correlation with the strongest correlation shown in (B).

FIGURE 3.

Subregions of the transcriptome show changes in reactivity across four temperatures. Violin plots overlayed with box plots (A–C) and scatter plots (D–F) are formatted as in Figure 2. Mean reactivity across four temperatures for (A) 5′ UTR, (B) CDS, and (C) 3′ UTR. Global trends in mean reactivity exhibited by the entire transcriptome (Fig. 2) are similar to those displayed in the CDS plot (B). (D–F) Scatter plots showing Δ reactivity against Δ abundance of 23°C–30°C (left), 30°C–37°C (middle), and 37°C–42°C (right) for (D) 5′ UTR, (E) CDS, and (F) 3′ UTR. Trends exhibited by the entire transcriptome are likewise displayed in the CDS plots (E).

FIGURE 4.

Mapping in vivo reactivity onto a known RNA motif. The tyrS T box riboswitch from B. subtilis contains a k-turn in the lower portion of the structure (PDB ID: 2KZL). This particular k-turn is characterized by three bulged A residues followed by two GA base pairs. The bulged A residues show moderate (orange) to strong (dark orange) DMS reactivity at 37°C.

FIGURE 5.

Reactivity changes with temperature identify potential regulatory RNA elements. (A) Reactivity per nucleotide for the glpF 5′ UTR at 23°C, 30°C, 37°C, and 42°C. Numbering extends from the start of transcription to the start codon. (B) Reactivity for nucleotides 88–150 of the glpF 5′ UTR mapped onto the predicted secondary structure. Normalized reactivity ranges from low (pale orange) to high (dark orange). The position of the SD sequence is shown. (C) Reactivity per nucleotide for the glpT 5′ UTR at 23°C, 30°C, 37°C, and 42°C. Numbering extends from the start of transcription to the start codon. (D) Reactivity for nucleotides 84–158 of the glpT 5′ UTR mapped onto the predicted secondary structure. Normalized reactivity ranges from low (pale orange) to high (dark orange). The position of the SD sequence is shown. The schematics in panels B and D begin immediately downstream from experimentally identified intrinsic terminators and extend to the AUG start codon (shown in lowercase letters).

FIGURE 6.

Effect of temperature on glpF expression. (A) Schematic representation of the glpF′-′bgaB translational fusion showing the promoter (P) and SD sequence. The glpF RNAT is depicted as a lollipop. (B) glpF RNAT. SD sequence is in magenta and two point mutations that stabilize the thermometer are in cyan. (C) β-Galactosidase activity of the glpF′-′bgaB translational fusion grown at the indicated temperatures during exponential and stationary phase growth. Values are averages of at least three independent experiments ± sd. WT, wild type fusion; mut, mutant fusion with the two mutations shown in (B).

FIGURE 7.

Effect of temperature on glpT expression. (A) Schematic representation of the glpT′-′bgaB translational fusion showing the P and SD sequence. The glpT RNAT is depicted as a lollipop. (B) glpT RNAT. SD sequence is in magenta and three point mutations that stabilize the thermometer are in cyan. (C) β-Galactosidase activity of the glpT′-′bgaB translational fusion grown at the indicated temperatures during exponential and stationary phase growth. Values are averages of at least three independent experiments ± sd. WT, wild type fusion; mut, mutant fusion with the three mutations shown in (B). (D) Same as (C) except the intrinsic terminator was deleted from the glpT leader region.

FIGURE 8.

Schematic representing possible model of regulation. At low temperatures, the 5′ UTR for both glpF and glpT showed little reactivity and are therefore depicted as folded and import is low (red). As temperature increases, the 5′ UTR reactivity increased and is depicted as melting from a structured RNA to a more unstructured RNA. This in turn increased expression and could result in increased import (green) of glycerol (G) and glycerol-3-phosphate (G3P) by GlpF and GlpT, respectively. These solutes could function as chemical chaperones to protect proteins during this temperature increase.

Elizabeth A. Jolley