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. 2019 Sep 10;10:2084.
doi: 10.3389/fmicb.2019.02084. eCollection 2019.

YtrA Sa, a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius

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YtrA Sa, a GntR-Family Transcription Factor, Represses Two Genetic Loci Encoding Membrane Proteins in Sulfolobus acidocaldarius

Liesbeth Lemmens et al. Front Microbiol. .
Free PMC article

Abstract

In bacteria, the GntR family is a widespread family of transcription factors responsible for the regulation of a myriad of biological processes. In contrast, despite their occurrence in archaea only a little information is available on the function of GntR-like transcription factors in this domain of life. The thermoacidophilic crenarchaeon Sulfolobus acidocaldarius harbors a GntR-like regulator belonging to the YtrA subfamily, encoded as the first gene in an operon with a second gene encoding a putative membrane protein. Here, we present a detailed characterization of this regulator, named YtrASa, with a focus on regulon determination and mechanistic analysis with regards to DNA binding. Genome-wide chromatin immunoprecipitation and transcriptome experiments, the latter employing a ytrA Sa overexpression strain, demonstrate that the regulator acts as a repressor on a very restricted regulon, consisting of only two targets including the operon encoding its own gene and a distinct genetic locus encoding another putative membrane protein. For both targets, a conserved 14-bp semi-palindromic binding motif was delineated that covers the transcriptional start site and that is surrounded by additional half-site motifs. The crystallographic structure of YtrASa was determined, revealing a compact dimeric structure in which the DNA-binding motifs are oriented ideally to enable a specific high-affinity interaction with the core binding motif. This study provides new insights into the functioning of a YtrA-like regulator in the archaeal domain of life.

Keywords: GntR; Sulfolobus; YtrA; archaea; membrane protein; transcription regulation.

Figures

FIGURE 1
FIGURE 1
Sulfolobales genomes encode a YtrA-like homolog. (A) Genomic organization of ytrA-like genes in Sulfolobales. Gene functions are mentioned based on annotations (Chan et al., 2012) with the following color code: blue, (putative) transcription regulators; orange, (putative) membrane proteins and/or transporters; green, (putative) metabolic enzymes; purple, (putative) transposon-associated functions. All Sulfolobales strains of which genome sequences are present in the SyntTax database (Oberto, 2013) harbor a ytrA homolog. The strains represented in the scheme are Sulfolobus acidocaldarius DSM639, Sulfolobus solfataricus P2, Sulfolobus islandicus REY15A and Acidianus manzaensis YN25. (B) Sequence alignment of YtrA-like GntR-family regulators in archaea and bacteria. Following sequences are shown in the alignment: Q4J7S4 Sulfolobus acidocaldarius (Sa), A0A1W6JWH0 Acidianus manzaensis (Am), Q97XX0 Sulfolobus solfataricus (Ss), F0NDQ3 Sulfolobus islandicus (Si), Q9HK68 Thermoplasma acidophilum (Ta), O34712 Bacillus subtilis (Bs), Q9XA65 Streptomyces coelicolor (Sc), Q8NLJ5 Corynebacterium glutamicum (Cg). Conserved amino acid residues are indicated in shades of green. Sequence identities are provided next to each sequence. Position numbers are indicated with respect to YtrASa, while secondary structure elements are displayed based on the crystal structure of the C. glutamicum YtrA (PDB:2EK5) (Gao et al., 2007). A red asterisk denotes an Arg that is presumed to play an important role in DNA binding.
FIGURE 2
FIGURE 2
YtrASa interacts with two genomic loci in vivo. (A) Zoomed images of two sections of the genomic binding profile of YtrASa as monitored by ChIP-seq, for which enrichment was observed. Below the profile, a schematic representation of the genomic organization of the in vivo binding regions is shown with the indication of the ChIP-seq peak summit locations (red triangle). The black curve represents the ChIP sample, while the red curve represents the mock experiment; cpm = counts per million. (B) Validation of ChIP enrichment using a qPCR approach with probes designed based on the detected binding region in ChIP-seq (targets are named after the gene located closest in the neighborhood). This experiment has been performed for biological triplicates, with the data shown being representative for all replicate experiments. Fold enrichment is expressed relative to a genomic region within the ORF of Saci_1336 shown not to be bound by YtrASa in the genome-wide ChIP-seq experiment.
FIGURE 3
FIGURE 3
YtrASa forms complexes with its DNA targets in vitro and with high affinity. Electrophoretic mobility shift assays (EMSAs) of YtrASa binding to radiolabeled DNA probes of about 100 bp representing the ChIP-seq peaks. Molar protein concentrations are indicated above the autoradiograph. Populations of free DNA (F), single-stranded DNA (ssDNA), YtrASa-bound DNA (B1 and B2) and non-specific DNA-protein complexes (NS) are indicated with an arrowhead or accolade. Below, schematic representations are shown of the location of the probe (indicated in red) used in EMSA with respect to the open reading frames (ORFs) in the genomic neighborhood of the target regions. Calculations of apparent dissociation constants (KDapp) and Hill coefficients (n) are based on densitometric analysis of free DNA bands followed by binding curve analysis.
FIGURE 4
FIGURE 4
YtrASa binds a conserved binding motif covering the transcriptional start for both targets. (A) Autoradiographs of DNase I footprinting experiments performed with 102- or 100-bp DNA probes having either the bottom (ytrASa target) or top (Saci_2078 target) strand 32P-labeled, respectively. Arrows indicate the direction of electrophoresis; “A + G” and “C + T” represent the purine- and pyrimidine-specific Maxam-Gilbert sequencing ladders, respectively; “F” denotes the population of free DNA; the other lanes show samples to which YtrASa protein was added at the following concentration range: 40–571 nM for the ytrASa target and 40–230 nM for the Saci_2078 target. Protected zones are indicated with a horizontal red line, while DNase I hypersensitivity sites are pointed out with ball-and-sticks symbols. Conserved binding motifs are boxed. Below each autoradiograph, the corresponding nucleotide sequence is shown with annotation of the observed protection zone (red letters) and hypersensitivity sites (ball-and-stick symbols). The translational start codon is indicated with an arrow below the sequence, while the transcription start site (TSS) is pointed out with an arrow above the sequence. The TSS of ytrASa is based on a high-resolution transcriptome database (Cohen et al., 2016), while that of Saci_2078 is predicted based on sequence alignment of both promoter regions. Putative factor B recognition element (BRE) and TATA box promoter elements are boxed. (B) Sequence logo representation of a nucleotide sequence alignment of both target promoter regions, with indication of BRE, TATA box, start codon (SC) and TSS.
FIGURE 5
FIGURE 5
Palindromic elements in the binding motif that support YtrASa interaction as shown by mutational analysis. (A) Sequence logo depicting a phylogenetic footprint using the forward and reverse sequences of the conserved YtrA binding motif identified in the ytrA promoter regions of S. acidocaldarius, S. solfataricus, S. islandicus and A. manzaensis and in the Saci_2078 promoter region of S. acidocaldarius. Primary (prim) and secondary (sec) binding motif elements are indicated. The center of dyad symmetry is annotated with a yellow oval symbol. (B) Overview of the experimental design of mutated binding site variants (MUT), with the wild type (WT) binding site of the ytrASa target as a starting point. Palindromic residues are depicted in bold and mutated residues in red. (C) Graphical representation of binding curve analysis based on densitometric analysis of electrophoretic mobility shift assays performed with 50-bp DNA probes containing binding site variants as presented in panel B. Averages of calculated apparent equilibrium dissociation constants (KDapp) are shown as well for each of the variants. Experiments were performed in duplicate. KDapp discrepancies for the 50-bp WT probe used in this experiment and the 102-bp probe used in the initial EMSA experiment shown in Figure 3 can be explained by the differing length, which affects binding affinity (Peeters et al., 2007).
FIGURE 6
FIGURE 6
YtrASa has a dimeric structure with a distance of 36 Å between the DNA-binding domains. (A) Crystallographic structure of the YtrASa dimer with indication of the different chains in different colors (chain A: α1-5; chain B: α1-4) and the distance between two corresponding α2 residues (Arg42). The α5 helix of chain B could not be modeled due to poor electron density. In the inset, the protein structure of the YtrA-like CGL2947 of Corynebacterium glutamicum is displayed (PDB:2EK5) (Gao et al., 2007) with indication of the distance between conserved α2 residues. (B) Structural alignment of chains A and B of YtrASa dimer with indication of the different domains.
FIGURE 7
FIGURE 7
YtrASa exerts repression on its direct target genes encoding two membrane proteins. A bar plot shows relative gene expression levels as determined by RT-qPCR, comparing a ytrASa overexpression strain with its isogenic wild type. Error bars represent the standard deviations of three biological repeats. Average fold-change values of RNA-Seq analysis are depicted by a diamond. Gray lines represent a fold change of 2 and 0.5, respectively.
FIGURE 8
FIGURE 8
Hypothetical model of protein-DNA interactions that are possibly established when a YtrASa dimer binds the primary consensus binding motif. This model has been built using Pymol (DeLano, 2002), with indication of the consensus bp and the wHTH helices α2 and α3. Two conserved residues presumed to be important for DNA binding (Suvorova et al., 2015), Arg42 and Asn51, are depicted in a stick representation. The Arg42 residues and GC/CG bp that are hypothesized to interact and to be essential for sequence-specific interaction are colored red.

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