RNA-binding proteins stabilize antiterminator structures.

<p>Alternative stem-loop structures in transcribed leader regions from representative operons are shown. Terminator stem regions that participate in alternative structures are in blue, whereas antiterminator regions are in red. (A) NasR-responsive <i>nasF</i> operon leader in termination conformation. Dots indicate the critical residues A1 and G4 in the P1 and P2 loops. (B) Each ANTAR monomer is hypothesized to bind one of the loops, P1 and P2. Stabilizing the P2 antiterminator structure permits transcription readthrough <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002773#pgen.1002773-Ramesh1" target="_blank">[6]</a> by shortening the terminator stem and separating it from the poly-U tract <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002773#pgen.1002773-Peters1" target="_blank">[2]</a>. (C) LicT-responsive <i>bglP</i> operon leader in termination conformation. RAT is the ribonucleic antiterminator. (D) Each CAT (co-antiterminator) monomer binds to the antiterminator stem. Unusual base-pairing within the antiterminator stem is depicted schematically <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002773#pgen.1002773-Yang1" target="_blank">[25]</a>. Stablilizing the antiterminator structure permits transcription readthrough <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002773#pgen.1002773-Houman1" target="_blank">[22]</a> by shortening the terminator stem and separating it from the poly-U tract.</p

Genetic network of HMG-box genes that regulate mating in <i>P. anserina</i>.

<p>Arrows with heads and blunt ends indicate activation and repression, respectively. The numbers next to the arrows indicate the average FC in gene expression between the wild-type and mutant strains.The relationship between <i>mtHMG1</i> and <i>PaHMG5</i> may be mediated by <i>KEF1</i>. Alternatively, <i>mtHMG1</i> may by-pass <i>KEF1</i> to repress <i>PaHMG5</i> directly or indirectly. The consistency in the FCs suggest that <i>PaHMG6</i> by-passes this cascade to activate <i>PaHMG5</i> and <i>PaHMG8</i> either directly or indirectly. The numbers next to the arrows connecting the mating-type genes (<i>FMR1</i> and <i>FPR1</i>) and the downstream target genes are FCs that were obtained from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Bidard1" target="_blank">[73]</a>.</p

Selection of binders from the αRep libraries.

a<p>The libraries indicated were used for the selections against the corresponding targets.</p>b<p>The phage-ELISA results are indicated as the number of clones giving a positive signal <i>versus</i> the number of clones tested. A Clone was scored as positive if its measured signal/noise <i>ratio</i> was greater than five.</p>c<p>The sequences were determined among the phage-ELISA positive clones. For each target, the number of distinct sequences is indicated over the total number of sequences determined.</p>d<p>The soluble expression of phage-ELISA positive clones was probed using CoFi blot or Western blot experiments after liquid expression cultures. Reported <i>ratios</i> indicate the number soluble proteins over the number of clones tested.</p>e<p>The properties of the clones used for further characterization were determined by ITC, DSC and/or SEC as described below. The number of internal repeats for each binder is indicated in parentheses.</p

Biophysical characterization of the bGFP-A and GFP/bGFP-A complex.

<p>(A) ITC calorimetric titrations. Concentrations values are expressed in monomer concentrations. (▪): Tiration of GFP (35 μM) with bGFP-A (350 μM). (□): The bGFP-A binding specificity was tested by titration with NCS-wt (bGFP-A 30 μM, NCS-wt 350 μM). (B) The GFP-binding specificity was evaluated by ITC analysis of injection of bA3–1 (360 μM) in a solution of GFP (30 μM). (C) Size Exclusion Chromatography (Superdex 75 10/300) of the selected bGFP-A and GFP. (▾): SEC Elution profile of a mixture of GFP (2.25 nmol) and the binder bGFP-A (6.75 nmol). (∇): elution profile of the bGFP-A alone (2.25 nmol). (<b></b>): elution profile of the GFP alone (6.75 nmol). (D) Affinity determination of selected bGFP-A using SPR. Different concentrations of bGFP-A (71,3; 118; 142,6; 237,6; 713; 1426 nM) were applied to flow cell with immobilized biotinylated EGFP for 120 s followed by washing buffer flow. The sensorgrams were corrected for non-specific binding by subtraction of a channel without EGFP bound (grey curve). The fits of k_on and k_off rates are indicated by black dashed line. K_d values were computed using k_off  = 1.7×10⁻⁴ s⁻¹ for all concentrations and k_on  = 4.3, 4, 2.2, 2.6, 2, 2×10⁴M⁻¹ s⁻¹ for the increasing concentrations respectively.</p

Alignment of HMG domains from the 12 HMG-box proteins of <i>P. anserina</i>.

<p>The alignment was performed using ClustalW2 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Larkin1" target="_blank">[105]</a> and colored according to the Clustal X color scheme provided by Jalview <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Waterhouse1" target="_blank">[106]</a>. This color scheme is displayed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642.s008" target="_blank">Table S1</a>.</p

Phenotypes of the <i>P. anserina</i> mutants with deleted HMG-box genes.

a<p>see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen-1003642-g004" target="_blank">Figure 4 A</a>.</p>b<p>see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen-1003642-g004" target="_blank">Figure 4 B</a>. ø: outside diameter of the ring in mm (mean of measurements made in eight independent cultures).</p

Expression of HMG-box genes and mating-type target genes in strains with HMG-box gene deletion.

<p>FCs from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642.s010" target="_blank">Table S3</a> to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642.s014" target="_blank">S7</a> were converted to a heat map using Matrix2png <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Pavlidis1" target="_blank">[108]</a>. Squares for FCs with non-significant statistical values are in black. Squares for inapplicable values are in white. (A) Heat map in the <i>mat+</i> strains. The FCs of HMG-box genes, <i>FPR1</i> and selected FPR1 target genes are represented as indicated on the scale for the following strains: <i>mat+ ΔPahmg6</i> (delPahmg6), <i>mat+ Δmthmg1</i> (delmthmg1), <i>mat+ Δkef1</i> (delkef1), <i>mat+ ΔPahmg5</i> (delPahmg5), and <i>mat+ ΔPahmg8</i> (delPahmg8). Gene number: <i>MFP</i>, Pa_2_2310; <i>PRE2</i>, Pa_4_1380; <i>AOX</i>, Pa_3_1710; <i>PEPCK</i>, Pa_4_3160. (B) Heat map in the <i>mat−</i> strains. The FCs of HMG-box genes, <i>FMR1</i> and selected FMR1 target genes were represented as indicated on the scale in (A) for the following strains: <i>mat− ΔPahmg6</i> (delPahmg6), <i>mat− Δmthmg1</i> (delmthmg1), <i>mat− Δkef1</i> (delkef1), <i>mat− ΔPahmg5</i> (delPahmg5), and <i>mat− ΔPahmg8</i> (delPahmg8). Gene number: <i>MFM</i>, Pa_1_8290; <i>PRE1</i>, Pa_7_9070.</p

Crosses of HMG-box gene deletion mutants.

<p>(A) Analysis of male and female fertility of HMG-box gene deletion mutants in crosses with wild-type tester strains. When cultures were confluent, sterile water was poured and dispersed over the surface of the mycelium. As each strain produced spermatia (male cells) and protoperithecia (female organs) regardless of its mating type, reciprocal fertilization of the mutant and wild-type strains took place and indicated whether the mutant was fertile as a male (donor) or as a female (receptor). For <i>ΔPahmg5</i> some perithecia differentiated at the contact zone where mutant and wild-type mycelia fuse. In the resulting heterokaryotic mycelium, wild-type nuclei complement the male and/or female sterility defect of the mutant. Those perithecia were fertile and expelled numerous asci allowing genetic analysis. (B) Analysis of perithecium distribution in homozygous crosses of HMG-box gene deletion mutants. Fragmented mycelium from <i>mat+</i> and <i>mat−</i> strains with the same HMG-box deletion were deposited at the center of a Petri dish and incubated until perithecia formed. Typically, the wild-type strains differentiated perithecia within a ring-like area.</p

Unrooted phylogram for the HMG-box superfamily.

<p>Clustering of core amino acid sequences using maximum-likelihood and model LG+G <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Le1" target="_blank">[107]</a>. Color labeling: MATα_HMG (A, green), MATA_HMG (B, yellow), SOX-TCF_HMG (C, orange), HMGB-UBF_HMG (D, H, blue), MAT1-1-3 in MATA_HMG (E, white) and STE11 in MATA_HMG (G, red). Other labels: Microsporidia MAT sex locus in HMGB-UBF_HMG (F, grey), <i>Phycomyces blakesleeanus</i> (Zygomycota) sexM (Phybl8) and sexP (Phybl9) (purple) and <i>P. anserina</i> proteins (red dots). LR-ELW values greater than 70% are shown. Abbreviations: <i>Ailuropoda melanoleuca</i> (Ailme); <i>Ajellomyces capsulatus</i> (Ajeca); <i>Alternaria alternata</i> (Altal); <i>Alternaria brassicicola</i> (Altbr); <i>Anopheles gambiae</i> (Anoga); <i>Antonospora locustae</i> (Antlo); <i>Arabidopsis thaliana</i> (Arath); <i>Aspergillus fumigatus</i> (Aspfu); <i>Aspergillus nidulans</i> (Aspni); <i>Botryotinia fuckeliana</i> (Botfu); <i>Bipolaris sacchari</i> (Bipsa); <i>Caenorhabditis elegans</i> (Caeel); <i>Candida albicans</i> (Canal); <i>Cervus elaphus yarkandensis</i> (Cerel); <i>Ciona savignyi</i> (Ciosa); <i>Cochliobolus heterostrophus</i> (Coche); <i>Cochliobolus homomorphus</i> (Cocho); <i>Coprinopsis cinerea</i> (Copci); <i>Cryphonectria parasitica</i> (Crypa); <i>Culex quinquefasciatus</i> (Culqu); <i>Danio rerio</i> (Danre); <i>Dothistroma pini</i> (Dotpi); <i>Drosophila melanogaster</i> (Drome); <i>Enterocytozoon bieneusi</i> (Entbi); <i>Encephalitozoon cuniculi</i> (Enccu); <i>Fusarium acaciae-mearnsii</i> (Fusac); <i>Gibberella fujikuroi</i> (Gibfu); <i>Gibberella zeae</i> (Gibze); <i>Homo sapiens</i> (Homsa); <i>Lachancea thermotolerans</i> (Lacth); <i>Magnaporthe oryzae</i> (Magor); <i>Mycosphaerella graminicola</i> (Mycgr); <i>Podospora anserina</i> (Podan); <i>Neurospora crassa</i> (Neucr); <i>Penicillium marneffei</i> (Penma); <i>Phycomyces blakesleeanus</i> (Phybl); <i>Pneumocystis carinii</i> (Pneca); <i>Pyrenopeziza brassicae</i> (Pyrbr); <i>Pyrenophora teres</i> (Pyrte); <i>Rhynchosporium secalis</i> (Rhyse); <i>Saccharomyces cerevisiae</i> (Sacce); <i>Schizosaccharomyces japonicus</i> (Schja); <i>Schizosaccharomyces pombe</i> (Schpo); <i>Sordaria macrospora</i> (Sorma); <i>Stemphylium sarciniforme</i> (Stesa); <i>Strongylocentrotus purpuratus</i> (Strpu); <i>Takifugu rubripes</i> (Takru); <i>Ustilago maydis</i> (Ustma); <i>Verticillium dahliae</i> (Verda); <i>Xenopus laevis</i> (Xenla); and <i>Zygosaccharomyces rouxii</i> (Zygro). Numbers after species names indicate α1 proteins (1), MATA_HMG (2), MAT1-1-3 (3), SOX (4), HMGB-UBF_HMG (5) and other HMG domains (6–9). When more than one domain was present for the same species, the suffix a, b or c was used. Units indicate the number of amino acid changes per position. Species codes and accession numbers grouped by evolutionary affinity are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642.s009" target="_blank">Table S2</a>.</p

Conserved sequences found in the promoter region of HMG-box genes and mating-type target genes.

<p>(A) Conserved sequences from a number of HMG-box genes and mating-type target genes identified using MEME program <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Bailey1" target="_blank">[78]</a>. Position −1 is the first nucleotide upstream of the translation initiation codon. (B) Weblogo of the consensus sequence generated by MEME (assembled from sequences listed in A). (C) The <i>P. anserina</i> consensus sequence aligned with the binding site of HMG-box proteins: MATa-1 (<i>N. crassa</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Philley1" target="_blank">[109]</a>, SpSte11 (<i>S. pombe</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Sugimoto1" target="_blank">[30]</a>, Prf2 (<i>U. maydis</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Hartmann1" target="_blank">[38]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Urban1" target="_blank">[39]</a> and Mat2 (<i>C. neoformans</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003642#pgen.1003642-Kruzel1" target="_blank">[41]</a>.</p