<b>Effects of mutations in the </b><b><i>trp</i></b><b> attenuator on TRAP-mediated transcription termination </b><b><i>in vivo</i></b>.

<p><i>^a</i> − cells grown in the absence of exogenous L-tryptophan; +, cells grown in the presence of 50 µg/ml of L-tryptophan.</p><p><i>^b</i> Values are the average of two or three independent experiments, each performed in triplicate, ± the standard deviation.</p><p><i>^c</i> To determine fold regulation by TRAP, the ratio of BG4233 Trp- to BG2087 Trp+ was determined for each experiment, and the value shown is the average +/− the standard deviation.</p><p><i>^d</i> Fold tryptophan regulation was determined by taking the ratio of BG2087 Trp- to BG2087 Trp+ for each experiment, and the value shown is the average +/− the standard deviation.</p

Blocking transcription elongation with EcoRI* to provide time for TRAP binding.

<p><b>(A)</b> Diagram of EcoRI* blocking the transcription elongation complex. Cleavage defective E111Q EcoRI (EcoRI*) binds to its recognition site on the DNA template and blocks elongation of RNAP approximately 12–13 bp upstream of the first G of the GAATTC recognition site. EcoRI* is shown as an oval shape bound to its recognition site starting at +116 in the <i>trp</i> leader region. RNAP is shown as a shaded grey shape. The nascent RNA is shown in bold with the 3′-most 8 residues paired with the template DNA. The last 4 (G/U)AG repeats of the TRAP binding site are circled. <b>(B)</b> Gel electrophoresis using a 6% polyacrylamide-8M urea gel analysis of block-and-release assay for the wild type (WT), U-stretch Disruption (US), C125G mismatch stem (MM), and disrupted stem (DS) attenuator templates. EcoRI* was allowed to bind to the DNA template prior to initiating transcription, TRAP was added, and transcription was then allowed to proceed until the TECs were blocked by EcoRI*. EcoRI* was then dissociated from the DNA by addition of 0.5 M KCl, allowing transcription to resume. The location of transcripts from blocked TECs (B), terminated at the attenuator (T), and read through (RT) transcripts are indicated on both sides of the gel. The percentage of transcription termination is displayed below the lane numbers (%T).</p

Effects of reduced NTP levels and of NusA on TRAP-mediated transcription termination <i>in vitro</i>.

<p>Polyacrylamide gel electrophoresis analysis of <i>in vitro</i> transcription of templates with the Wild Type (WT), U-stretch (US), mismatch stem (MM), and disrupted stem (DS) <i>trp</i> attenuator regions. Transcription reactions were performed in the absence or presence of 0.5 µM TRAP, and 0–1.5 µM NusA. The positions of read through (RT) and terminated (T) transcripts are indicated at the left. The percentage termination (%T) for each reaction is at the bottom of each lane.</p

<i>In vitro</i> transcription attenuation assays.

<p><b>(A)</b> Diagram of DNA templates used for <i>in vitro</i> transcription attenuation assays. The two major transcripts produced from these templates include read through (RT; ∼140 nts) and terminated (T: ∼320 nts) transcripts are shown below the diagram of the template. <b>(B)</b> 6% polyacrylamide-8M urea gel electrophoresis analysis of the products of <i>in vitro</i> transcription of the wild type (WT), U-stretch (US), mismatch stem (MM), and disrupted stem (DS) templates in the absence and presence of 0.25 or 0.5 µM TRAP. Positions of read through (RT = 320 nt) and terminated (T = 140 nt) transcripts are indicated at left side of the figure. The percentage of transcripts terminating at the attenuator (%T) for each reaction is shown at the bottom of each lane.</p

<i>Trp</i> attenuator regions of different bacterial species.

<p>Predicted attenuator RNA structures from the <i>trp</i> leader region of several TRAP containing bacterial species using M-fold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088097#pone.0088097-Zuker1" target="_blank">[46]</a>. The last repeat in the proposed TRAP binding is circled and the predicted attenuator structure is shown as well as 20 residues downstream of the predicted base-paired stem.</p

Mapping the site of TRAP-mediated transcription termination <i>in vivo</i>.

<p><b>(A)</b> Schematic diagram of RNase protection assays (RPA) using ³²P-labeled antisense probes and cellular RNA from <i>B. subtilis</i>. Antisense RNA probes are indicated as dotted lines and cellular RNA is displayed as solid lines (thick line for WT <i>trp</i> RNA and thin line for RNA from the mutant <i>trpE′-′lacZ</i> fusions). The WT antisense probe was hybridized to WT cellular RNA, and the mutant antisense probes, U-stretch (US) mutant or Disrupted stem (DS), were hybridized to RNA from <i>B. subtilis</i> containing mutant <i>trpE′-′lacZ</i> fusions, as well as to RNA from WT cells. The predicted lengths of the protected products are shown at the right side of the figure. <b>(B)</b> RNase protection assays to determine location of transcription termination in <i>trp</i> leader regions containing mutant attenuators. WT and mutant <i>trp</i> antisense RNA (Probes) were incubated with cellular RNA from WT <i>B.subtilis</i> or strains containing <i>trpE′-′lacZ</i> fusions with either the U-stretch (US) mutant or Disrupted stem (DS) mutant attenuator prior to digestion with RNaseT1/A. Black arrow represents the location of transcripts terminated at the WT attenuator; (*) indicates bands that are from probes designed to hybridize with transcripts terminated at mutant attenuator present in <i>trpE′-′lacZ</i> fusion when they pair with native WT <i>trp</i> leader transcripts terminated at the WT attenuator. Reactions were run on 8% denaturing polyacrylamide gels. Markers were generated by T7 RNAP run off transcription of templates that contain an XbaI site from +133–138, or a PstI site from +134–139 relative to the start of transcription respectively. In each case a T7 promoter was placed upstream of the template so that transcription initiation occurs at +1. The 137 nt RNA marker was generated by transcription of the XbaI cleaved template, and the 134 nt RNA marker was from template cut with PstI.</p

<b>Effects of deletions within the </b><b><i>trp</i></b><b> leader region on TRAP-mediated transcription termination </b><b><i>in vivo</i></b>.

<p><i>^a</i> − cells grown in the absence of exogenous L-tryptophan; +, cells grown in the presence of 50 µg/ml of L-tryptophan.</p><p><i>^b</i> Values are the average of two or three independent experiments, each performed in triplicate, ± the standard deviation.</p><p><i>^c</i> To determine fold regulation by TRAP, the ratio of BG4233 Trp- to BG2087 Trp+ was determined for each experiment, and the value shown is the average +/− the standard deviation.</p><p><i>^d</i> Fold tryptophan regulation was determined by taking the ratio of BG2087 Trp- to BG2087 Trp+ for each experiment, and the value shown is the average +/− the standard deviation.</p

Substitutions in the stem-loop and U-stretch of the <i>trp</i> attenuator.

<p><b>(A)</b> Diagram depicting the WT and mutant <i>trp</i> attenuators with altered bases in the stem-loop structure or U-stretch. Substitutions are indicated in bold and are underlined. Numbers adjacent to structure names indicate fold TRAP regulation observed <i>in vivo</i> using β-galactosidase assays (results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088097#pone-0088097-t001" target="_blank">Table 1</a>). <b>(B)</b> Schematic representation of the transcriptional reporter fusions with <i>lacZ</i> that were used to examine TRAP-mediated regulation of transcription termination <i>in vivo</i>. The arrow represents the <i>trp</i> promoter and <i>trpL</i> refers to the regulatory leader region (See Methods for details about each gene fusion). Each <i>trpE′-′lacZ</i> fusion was integrated into the <i>B. subtilis</i> genome at the <i>amyE</i> locus.</p

Model of transcription attenuation of the <i>B. subtilis trp</i> operon.

<p>Bold black letters designate the complementary strands of the attenuator (C/D) (highlighted in blue) and antiterminator (A/B) RNA structures. TRAP is shown in a ribbon diagram with each subunit in a different color. The 11 (G/U)AG repeats of the TRAP binding site are circled and numbered in green. Small black numbers indicate RNA residues relative to the start of transcription. When tryptophan is limiting, the AB antiterminator RNA structure forms, allowing read through of the <i>trp</i> operon. In excess tryptophan, TRAP binds to the nascent RNA and prevents formation of the antiterminator structure, which allows formation of the attenuator, leading to transcription termination.</p

Solution NMR Experiment for Measurement of ¹⁵N–¹H Residual Dipolar Couplings in Large Proteins and Supramolecular Complexes

NMR residual dipolar couplings (RDCs) are exquisite probes of protein structure and dynamics. A new solution NMR experiment named 2D SE2 <i>J</i>-TROSY is presented to measure N–H RDCs for proteins and supramolecular complexes in excess of 200 kDa. This enables validation and refinement of their X-ray crystal and solution NMR structures and the characterization of structural and dynamic changes occurring upon complex formation. Accurate N–H RDCs were measured at 750 MHz ¹H resonance frequency for 11-mer 93 kDa ²H,¹⁵N-labeled Trp RNA-binding attenuator protein tumbling with a correlation time τ_c of 120 ns. This is about twice as long as that for the most slowly tumbling system, for which N–H RDCs could be measured, so far, and corresponds to molecular weights of ∼200 kDa at 25 °C. Furthermore, due to the robustness of SE2 <i>J</i>-TROSY with respect to residual ¹H density from exchangeable protons, increased sensitivity at ¹H resonance frequencies around 1 GHz promises to enable N–H RDC measurement for even larger systems