TraR, a homolog of a RNAP secondary channel interactor, modulates transcription

Abstract

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.

Figure 1. Sequence Alignment Between DksA and TraR.

(A) Alignment of TraR sequence with DksA. TraR secondary structure prediction was performed with the PSIPRED prediction method and is labeled below the TraR sequence. H = helix, E = strand, and C = loose coil. PSIPRED predicts a high probability of an initial helix present in TraR. (B) Model of DksA highlighting (red shading) residues aligned between TraR and DksA.

Figure 2. Activation of the livJ Promoter by Expression of TraR.

β-galactosidase activity of the P_livJ-lacZ promoter fusion in LB media. Differential activity of β-galactosidase plotted. (A) Wild-type background. Induced (0.1 mM IPTG) expression of the TraR (triangles) activates the livJ promoter. WT DksA plasmid (squares) and control plasmid (circles). Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼520, 480, and 1010 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range. (B) As in A, but a ΔdksA background. Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼520, 670, and 1500 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range.

Figure 3. Inhibition of the rrnB P1 Promoter by Ectopically Expressed TraR.

Differential β-galactosidase activity of the rrnB P1-lacZ promoter fusion in LB media. (A,B) Wild-type background. (A) Effects of uninduced ectopic expression of TraR (open triangles) or DksA (open squares) on rrnB P1 activity. Plasmid control indicated by open circles. Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼100, 100, and 84 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range. (B) Induced (0.1 mM IPTG) ectopic expression of TraR (triangles) or DksA (squares) represses rrnB P1 activity. Circles, plasmid control. Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼95, 67, and 2.0 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range. (C,D) ΔdksA background. (C) Repression of β-galactosidase activity of rrnB P1-lacZ from uninduced ectopic expression of TraR (open triangles) or DksA (open squares) in the ΔdksA cells. Plasmid control indicated by open circles. Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼140, 84, and 88 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range. (D) Induced (0.1 mM IPTG) ectopic expression of TraR (triangles) or DksA (squares) represses rrnB P1 activity in ΔdksA. Circles, plasmid control. Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼140, 80, and 3.3 β-gal activity/OD₆₀₀ for pControl, pDksA, and pTraR, respectively. R² values>0.95 in this linear range. (E) TraR does not affect the P_lac promoter. β-galactosidase activity assays of the wild-type lac operon. TraR (triangles) induced (0.1 mM IPTG) from a multi-copy plasmid. Control plasmid indicated by circles.

Figure 4. TraR Inhibits Growth and Is Expressed Less than DksA.

(A,B) TraR Inhibits Growth. (A) Semilog plot of OD₆₀₀ vs. growth time resulting from induced (0.1 mM IPTG) expression of TraR (triangles) or DksA (squares) in the WT rrnB P1-lacZ background grown in LB-ampicillin at 32°C. Circles, plasmid control. (B) Figure depicting increasing doubling times (early logarithmic growth) resulting from successive dilutions in LB containing IPTG of a logarithmic culture induced for TraR (triangle). pControl and pDksA do not inhibit growth when treated similarly (35, 33, 35 minutes and 36, 36, 37 minutes were the respective doubling times, see Figure S2, Supplemental Materials). (C) Steady state levels of TraR expressed from pTraR-His₆ are lower than corresponding DksA levels. (Left) Protein extracts from indicated plasmids (±IPTG) were separated by SDS-PAGE (12%) and detected by Coomassie staining (upper left) or Western blotting using an anti-His₆ antibody (lower left). Bands corresponding to TraR and DksA are indicated by asterisks. (Right) TraR-His₆ and DksA-His₆ were pulled-down from identical amounts of protein extracts made after two hours of induction, subjected to SDS-PAGE, and detected by Coomassie blue staining (upper right) or Western blotting (anti-His₆ antibody) (lower right). Purification plasmid pET24a-TraR-His₆ was used as a positive control to visualize TraR-His₆ in both assays.

Figure 5. TraR Functions In Vivo Without ppGpp.

(A) Strong activation of β-galactosidase activity from P_livJ-lacZ in LB media with induced (0.1 mM IPTG) expression of TraR (triangles) in ΔrelA ΔspoT (ppGpp⁰) cells. DksA and control plasmids indicated by squares and circles, respectively. The ppGpp-independent activation is dependent on the 6^th Asp residue of TraR mutated in pTraR-D6N (X). Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼140, 400, 1900, and 150 β-gal activity/OD₆₀₀ for pControl, pDksA, pTraR, and pTraR-D6N, respectively. R² values>0.95 in this linear range. (B) Inhibition of β-galactosidase activity from rrnB P1-lacZ in LB media with induced (0.1 mM IPTG) expression of TraR (triangles) in a ΔrelA ΔspoT (ppGpp⁰) background. DksA and control plasmids indicated by squares and circles, respectively. Repression by TraR is abolished by mutation of 6^th Asp residue (pTraR-D6N, diamonds). Rates of β-galactosidase synthesis in exponential phase (from OD₆₀₀ 0.2 to 0.6) are ∼82, 120, 0.0, and 110 β-gal activity/OD₆₀₀ for pControl, pDksA, pTraR, and pTraR-D6N respectively. R² values>0.95 in this linear range. (C) Ectopic uninduced expression of DksA or TraR suppress the cell motility defects of either ΔdksA or ΔrelA ΔspoT (ppGpp⁰) ΔdksA cells. Strains were inoculated on low agar plates (0.375%) and grown for ∼24 hours at room temperature, at which the diameters of resulting growth areas produced by motile cells were measured (see Figure S3 in Supplemental Materials for representative picture). Means±standard deviation plotted.

Figure 6. TraR Inhibits rrnB P1 Transcription In Vitro Independent of ppGpp.

Single round transcriptions were performed in the presence (filled symbols) or absence (open symbols) of 250 µM ppGpp with increasing concentrations (0–600 nM) of TraR-His₆ (triangles) or DksA-His₆ (squares). Amounts of RNA were measured by phosphorimaging (A) and are quantified in (B), normalized to the equivalent units observed at TraR = 0 or DksA = 0 for each set of reactions separately. Means of 3 independent determinations of TraR (±standard deviation) and a DksA control experiment performed in parallel are plotted. In vitro DksA results agree with previously published data ,.