Effect of the 5′ terminal stem–loop structure on the affinity of the 5′-UTR for Hfq and ribosomes

<p><b>Copyright information:</b></p><p>Taken from "Both RNase E and RNase III control the stability of mRNA upon translational inhibition by the small regulatory RNA RyhB"</p><p>Nucleic Acids Research 2005;33(5):1678-1689.</p><p>Published online 21 Mar 2005</p><p>PMCID:PMC1069011.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () 5′ end-labelled and RNAs were incubated alone (lanes 1 and 6) or with increasing amounts (2-, 4-, 6- and 8-fold molar excess) of Hfq-hexamer (lanes 2–5 and 7–10, respectively), and the resulting mixtures were then analysed on a 6% native gel. The positions of free and RNAs as well as their complexes with Hfq (single and double asterisks, respectively) are indicated. () Differential decrease of 30S ribosome binding to mRNA by and competitor RNA, respectively. The RNA pre-annealed to the 5′ end-labelled primer was incubated with 30S ribosomal subunits in the presence of increasing amounts (1-, 2-, 4- and 8-fold molar excess) of competitor RNAs ( and , respectively). Translation inhibition complex formation was further analysed by primer extension as described in Materials and Methods. () Relative toeprints obtained on RNA [see (B)] using and RNA as competitors, respectively. The relative toeprints (%) were calculated as described by Hartz . () after quantitation of the toeprint and extension signals using the equation: [toeprint signal/(toeprint signal + extension signal)]

Impact of Hfq on the <i>Bacillus subtilis</i> Transcriptome

<div><p>The RNA chaperone Hfq acts as a central player in post-transcriptional gene regulation in several Gram-negative Bacteria, whereas comparatively little is known about its role in Gram-positive Bacteria. Here, we studied the function of Hfq in <i>Bacillus subtilis</i>, and show that it confers a survival advantage. A comparative transcriptome analysis revealed mRNAs with a differential abundance that are governed by the ResD-ResE system required for aerobic and anaerobic respiration. Expression of <i>resD</i> was found to be up-regulated in the <i>hfq⁻</i> strain. Furthermore, several genes of the GerE and ComK regulons were de-regulated in the <i>hfq⁻</i> background. Surprisingly, only six out of >100 known and predicted small RNAs (sRNAs) showed altered abundance in the absence of Hfq. Moreover, Hfq positively affected the transcript abundance of genes encoding type I toxin-antitoxin systems. Taken the moderate effect on sRNA levels and mRNAs together, it seems rather unlikely that Hfq plays a central role in RNA transactions in <i>Bacillus subtilis</i>.</p></div

The abundance of putative sRNA regulators of <i>resA</i> is independent of Hfq.

<p>(A) Partial sequence of the 5′-untranslated region of <i>resA</i> and of the initial coding region. The −10 and −35 regions of the <i>resA</i> operon promoter are highlighted in blue. Arrow: start site and direction of transcription. The start codon (ATG) of <i>resA</i> is indicated in red. Predicted base-pairing interactions of sequences of FsrA, SurA and sRNA 13 with the rbs of <i>resA</i> mRNA. (B) The levels of FsrA, SurA, sRNA 13 and 5S rRNA (loading control) were determined by Northern-blot analyses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098661#s3" target="_blank">Materials and Methods</a>. <i>Bacillus subtilis</i> strains 168 wt (wt) and 168Δ<i>hfq</i> (Δ) were grown in CS-glucose medium at 37°C. Samples for RNA extraction were withdrawn at the OD₆₀₀ values indicated on top. Only the relevant sections of the autoradiographs are shown.</p

DEseq analysis of <i>B. subtilis</i> 168 wt versus 168Δ<i>hfq</i>.

<p>(A) Differential abundance of transcripts in strain 168 wt and in strain 168Δ<i>hfq</i> during logarithmic growth (OD₆₀₀ = 0.7) (B) Differential abundance of transcripts in strain 168 wt and in strain 168Δ<i>hfq</i> in early stationary phase (OD₆₀₀ = 2.0). Each dot represents one transcript. The log2 fold-change is plotted against the mean expression level for each transcript. Red dots represent transcripts whose abundance is significantly altered (p-value adjusted for multiple testing <0.1).</p

Expression of the <i>hfq</i> gene and requirement of Hfq for survival of <i>B. subtilis</i>.

<p>(A) <i>Bacillus subtilis</i> strain 168 wt and GP1067 were grown in CS-glucose medium at 37°C. The <i>hfq</i> mRNA and 5S rRNA (internal control) levels were assessed by primer extension analyses in <i>B. subtilis</i> strain 168 (upper two panels). Strain GP1067 was used to monitor Hfq and ribosomal S2 (internal control) levels by quantitative western-blotting (lower two panels). Samples for RNA extraction and for western-blot analyses were withdrawn throughout growth at the OD₆₀₀ values indicated on top. Only the relevant sections of the autoradiographs and immunoblots are shown. (B) The <i>B. subtilis</i> strains 168 wt (triangles) and 168Δ<i>hfq</i> (squares) were co-cultivated in CS-glucose medium. Likewise, <i>B. subtilis</i> strains GP1067 (diamonds) and 168Δ<i>hfq</i> (circles) were co-cultivated in CS-glucose medium. Growth was monitored over 10 days by scoring the CFU. Immunodetection of Hfq-Flag (lower panel) in strain GP1067 was performed at the days indicated. Only the relevant section of the immunoblot is shown.</p

Down-regulation of <i>resA/D</i> expression in the presence of Hfq.

<p>(A) Fluorescence conferred by plasmid pADresAts (1) borne transcriptional <i>resA-gfp</i> fusion and by the plasmid pADresAtl (2) borne translational <i>resA::gfp</i> fusion, respectively, was determined in <i>B. subtilis</i> strains 168 wt (black bars) and 168Δ<i>hfq</i> (gray bars). (B) Fluorescence conferred by the plasmid pADresDts (1) borne transcriptional <i>resD-gfp</i> fusion and by the plasmid pADresDtl (2) borne translational <i>resD::gfp</i> fusion, respectively, was determined in <i>B. subtilis</i> strains 168 wt (black bars) and 168Δ<i>hfq</i> (gray bars). Error bars represent standard deviations.</p

Interactions of adenines in Hfq-nucleotide complexes.

<p>(<b>A</b>) Hydrogen bond <b>i</b>nteraction between ribose 2′-OH with the Gly-29 carbonyl atom, for which a specific main chain conformation is required. ATP is shown as balls and sticks with the following color code for atoms: C – yellow; N - blue; O - red; P – orange. Hfq₆₅ residues involved in the interaction are presented by a stick. (<b>B</b>) Superposition of Hfq_Bs-AGAGAG (<i>B. subtilis</i> Hfq), Hfq-ADP (<i>E. coli</i> Hfq), Hfq-polyA (<i>E. coli</i> Hfq), Hfq_Pa-ADPNP (<i>P. aeruginosa</i> Hfq) and Hfq₆₅-ATP complexes to highlight the spread of purine ring orientations around the normal to the ring. Blue and red circles enclose two main clusters: in the first the exocyclic N6 atom hydrogen bonds to the Gln-52′ side-chain, in the second to the Thr-61 side-chain. N6 atoms are depicted as spheres. The colour code for atoms in the first cluster is: C - cyan; N - blue; O - red; P – orange; in the second C - yellow, and in the outlier ligand of the Hfq_Pa-ADPNP complex C - green.</p

RNA annealing activity of Hfq is ATP-independent.

<p>(<b>A</b>) 5 nM of two complementary RNAs (Cy5–21R⁺, Cy3–21R⁻) were annealed in a microplate reader in the absence or presence of Hfq at 37°C in 50 mM Tris-HCl, 3 mM MgCl₂ and 1 mM DTT in the presence of different ATP concentrations. The donor (Cy3) and acceptor (Cy5) fluorescence emissions were quantified every second. The FRET index was calculated as F_Cy5/F_Cy3. The curves were least-square fitted with the second-order reaction equation for equimolar initial reactant concentrations: P_t = P·(1−1/(<i>k</i>_obs·t+1)); P_t = fraction annealed, P = max. FRET index. Note that the amplitude of the FRET index is only indicative and does not correspond to the absolute percentage of double-stranded RNAs. (<b>B</b>) Reaction constants for RNA annealing at different ATP concentrations.</p

The purine binding site between adjacent subunits on the distal face of <i>E. coli</i> Hfq.

<p>Side-chains of binding site residues are shown by a stick. A single ATP-molecule is depicted with the triple-phosphate protruding in the favored conformation (transparent orange stick). Inset: The solvent accessible area of Hfq hexamer, colored according to its electrostatic potential (red and blue correspond to negatively and positively charged residues, respectively), is shown from the distal face with four ATP molecules bound.</p