Bioinformatic identification of <i>T</i>. <i>thermophilus</i> HB8 promoters potentially regulated by SbtR.

<p>Shown are sequences +/- 200 bp of the first codon of a target gene identified through FIMO analysis as being potentially regulated by SbtR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159408#pone.0159408.t002" target="_blank">Table 2</a>). Longest open reading frames with identical orientation as the target gene are indicated with blue nucleotides. Open reading frames with opposite orientation are indicated with green nucleotides. Black nucleotides indicate intragenic regions. Potential promoter elements (-30 and -10 boxes, +1 start site of transcription) were identified using Softberry BPROM and are indicated with blue highlighting. SbtR-binding sites are indicated with yellow highlighting. Regions of overlap between SbtR-binding sites and promoter elements are indicated by green highlighting.</p

Validation of REPSA-selected SbtR-binding DNA species.

<p>Shown are LICOR images of electrophoretic mobility shift assays containing pooled DNA from either Round 1 (left lanes) or Round 7 (right lanes) of REPSA selection and different concentrations of SbtR protein (from left to right: 0, 1, 10, 100, or 1000 nM SbtR dimer). The electrophoretic mobility of two protein-DNA complexes (S2 and S1) as well as uncomplexed ST2R24 selection template (T) and IRD7_ST2R primer (P) are indicated at right of figure.</p

FIMO of Best Possible Match TGACTGGCCAGTCA.

<p>FIMO of Best Possible Match TGACTGGCCAGTCA.</p

Comparison of CASTing and REPSA selection methods.

<p>Shown are flow diagrams depicting Cyclic Amplification and Selection of Targets (CASTing) and Restriction Endonuclease Protection Selection and Amplification (REPSA), combinatorial selection methods for the identification of preferred ligand-binding sequences in duplex DNA. Both methods rely on large populations of randomized DNA sequences, ligand-binding to a subpopulation of these DNAs, and PCR amplification of selected DNAs. However, CASTing and similar methods rely on the physical separation of ligand-bound from unbound DNAs (<i>e</i>.<i>g</i>., immunoprecipitation) for its selection process, whereas REPSA utilizes ligand-dependent interference with a template inactivation process (type IIS restriction endonuclease cleavage) for selection.</p

Expression and purification of SbtR protein.

<p>Shown is a Coomassie Brilliant Blue R250-stained 4–20% SDS-PAGE gradient gel onto which was loaded whole cell extracts or partially purified fractions containing SbtR protein. Lanes shown left to right: (log) 14 μg whole cell extract from logarithmic growth <i>E coli</i> BL21(DE3) bacteria containing the plasmid pET-sbtR, (ind) 24 μg whole cell extract from the aforementioned bacteria following IPTG-induction, (pur) 36 μg purified SbtR protein loaded under standard reducing conditions, (pur/ox) as previous, except that the sample was loaded under oxidizing conditions. The location of molecular weight standards is indicated at the left of the figure. SbtR and SbtR₂ indicate the locations of reduced monomeric and oxidized dimeric SbtR proteins, respectively.</p

SbtR-binding to a consensus sequence as analyzed by biolayer interferometry.

<p>Shown are raw traces (dots) and best-fit lines generated by GraphPad Prism using data obtained from a fortéBIO Octet biolayer interferometer. Binding reactions contained (top to bottom) 150, 50, 16.7, 5.56, 1.85 nM SbtR dimer protein. (<b>A</b>) Target was biotinylated ST2 DNA containing the 14-bp consensus insert 5′-TGACTGGCCAGTCA-3′. (<b>B</b>) Biotinylated ST2 control DNA.</p