NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2′dG-sensing riboswitch from <i>Mesoplasma florum</i> by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation

Meta-analysis of normalized expression of differentially abundant transcripts under the conditions A-30 and B-96 when compared with condition P.

<p>The genes are grouped into the corresponding pathways (<a href="http://www.kegg.jp/kegg-bin/show_organism?org=pau" target="_blank">http://www.kegg.jp/kegg-bin/show_organism?org=pau</a>). For each group the overall behavior was summarized by the averaged log₂ fold change of its significantly modulated members. The column denotes A-30 versus P and B-96 versus P, respectively. The color code shown in the scale at the right denotes log₂-fold changes. Red indicates an overall decrease and green indicates an overall increase in the mRNA levels of genes in a particular pathway. The numbers of genes within each group are indicated by the numbers given in parenthesis.</p

Altered abundance of transcripts encoding antibiotic tolerance functions.

<p>Fold change under the condition B-96 when compared with P. Only ≥ ± 5 fold change was considered.</p

OprI is not required for susceptibility of PA14 towards the antimicrobial peptide SMAP-29.

<p><b>A)</b> Determination of the levels of <i>oprI</i> mRNA in P cells (P) and B-96 cells (B-96) by Northern-blot analysis. <i>In-vitro</i> transcribed <i>oprI</i> mRNA (0.5 ng) was used as a control (C). 5S rRNA served as a loading control. <b>B)</b> The strains were grown planktonically to an OD₆₀₀ of 2.0 in SCFM (P) and for 96 hours under anaerobic conditions (B-96). Then, the cultures were harvested and the β-galactosidase activities were determined. The bars depict β-galactosidase values conferred by the translational OprI-LacZ protein in strain PA14(pTLoprI) under the conditions P and B-96. The error bars represent standard deviations from three independent experiments. <b>C)</b> Susceptibility of PA14 (red) and PA14Δ<i>oprI</i> (green) towards the cationic peptide, SMAP-29, under aerobic conditions. The experiment was performed as outlined in Materials and Methods. <b>D)</b> Determination of the <i>oprI</i> mRNA levels by Northern-blot analysis in strains PA14 and PA14∆<i>oprI</i> at the time of addition of SMAP-29 to the cultures. <i>In vitro</i> transcribed <i>oprI</i> mRNA (1 ng) was used as a control (C). 5S rRNA served as a loading control.</p

MICs of different antibiotics for B-96 cells versus P cells.

<p>MICs of different antibiotics for B-96 cells versus P cells.</p

Biofilm formation of PA14 and transposon mutants thereof after anaerobic growth in SCFM medium after 96h.

<p>Biofilm formation was quantified by measuring the absorbance at 595 nm after crystal violet staining. The results are averaged from three independent experiments.</p