scholarly journals Bacterial promoter opening underpins ubiquitous transcriptional regulation by DNA supercoiling

2020 ◽  
Author(s):  
Raphaël Forquet ◽  
Maïwenn Pineau ◽  
William Nasser ◽  
Sylvie Reverchon ◽  
Sam Meyer
Keyword(s):  
Transcription Initiation ◽  
Environmental Changes ◽  
Double Helix ◽  
Transcriptional Regulator ◽  
Dna Supercoiling ◽  
Differential Response ◽  
Transcriptomic Response ◽  
Specific Step ◽  
Wide Range

AbstractDNA supercoiling acts as a basal transcriptional regulator, which contributes to the quick and global transcriptional response of bacteria to many environmental changes. In spite of this importance, mechanistic models explaining the differential response of promoters to global topological variations of the chromosome remain essentially lacking. Here, we present the first quantitative transcriptional regulatory model by DNA super-coiling, focusing on the specific step of promoter opening during transcription initiation. Based on the known physico-chemical properties of DNA denaturation, it involves only one global adjustable parameter and is yet able to predict the global supercoiling response of promoters in a wide range of bacteria, based on the sequence content of their “discriminator” element. We first show that it quantitatively predicts both in vitro and in vivo data from transcription assays focusing on individual model promoters. We then assess the universality of the mechanism by analyzing transcriptomes of phylogenetically distant bacteria under conditions of supercoiling variation: (1) by gyrase-inhibiting antibiotics, (2) by biologically relevant environmental stresses, (3) naturally acquired and inherited in the longest-running evolution experiment. The model robustly predicts a significant contribution of the entire transcriptomic response to transient or inherited supercoiling variations in various species. This study strongly suggests that the proposed physical mechanism is used as an ubiquitous regulatory mechanism in the whole prokaryotic kingdom, based on the fundamental mechanical properties of the double-helix.ImportanceDNA supercoiling acts as a global yet underestimated transcriptional regulator in bacteria. We propose the first quantitative model of this regulation mode, based on the specific step of promoter opening during transcription initiation, explaining the differential response of promoters to global topological variations of the chromosome. In contrast to classical mechanisms requiring dedicated regulatory molecules to bind target promoters, we show that global deformations of the DNA template itself underpin a selective response of each particular promoter, according to its “discriminator” sequence, by modulating the ability of RNA Polymerase to initiate transcription. This study defines the first systematic rule underpinning the ubiquitous regulatory action of DNA supercoiling on the core transcriptional machinery, in particular in response to quick environmental changes.

scholarly journals Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling

2019 ◽  
Author(s):  
Bilal El Houdaigui ◽  
Raphaël Forquet ◽  
Thomas Hindré ◽  
Dominique Schneider ◽  
William Nasser ◽  
...  
Keyword(s):  
Environmental Changes ◽  
Bacterial Genome ◽  
Transcriptional Regulator ◽  
Dna Supercoiling ◽  
Mechanistic Modeling ◽  
Mechanical Constraints ◽  
A Genome ◽  
Specific Regulation

AbstractDNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

scholarly journals Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling

2019 ◽  
Vol 47 (11) ◽  
pp. 5648-5657 ◽  
Author(s):  
Bilal El Houdaigui ◽  
Raphaël Forquet ◽  
Thomas Hindré ◽  
Dominique Schneider ◽  
William Nasser ◽  
...  
Keyword(s):  
Environmental Changes ◽  
Bacterial Genome ◽  
Transcriptional Regulator ◽  
Dna Supercoiling ◽  
Mechanistic Modeling ◽  
Mechanical Constraints ◽  
A Genome ◽  
Specific Regulation

Abstract DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

scholarly journals Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1192
Author(s):  
Francesco Tini ◽  
Giovanni Beccari ◽  
Gianpiero Marconi ◽  
Andrea Porceddu ◽  
Micheal Sulyok ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Fusarium Graminearum ◽  
Environmental Changes ◽  
Virulent Strain ◽  
Fungal Virulence ◽  
Wheat Seedlings ◽  
Wide Range ◽  
Conidia Production ◽  
Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

scholarly journals What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii

2021 ◽  
Author(s):  
Maiwenn Pineau ◽  
Shiny Martis B. ◽  
Raphael Forquet ◽  
Jessica Baude ◽  
Lucie Grand ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Intrinsic Property ◽  
Dna Supercoiling ◽  
Negative Bacterium ◽  
Genomic Context ◽  
Transcriptomic Response ◽  
Dickeya Dadantii ◽  
Gram Negative ◽  
Dna Relaxation

DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by gyrase inhibition were categorized as "supercoiling-sensitive". Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine, and obtained the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in their spatial and orientational organisation along the chromosome. Altogether, these results suggest that the "supercoiling-sensitivity" is not an intrinsic property of promoters, but depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after gyrase or topoisomerase I inhibition.

scholarly journals Transcriptional Regulation in the Hyperthermophilic ArchaeonPyrococcus furiosus: Coordinated Expression of Divergently Oriented Genes in Response to β-Linked Glucose Polymers

1999 ◽  
Vol 181 (12) ◽  
pp. 3777-3783 ◽  
Author(s):  
Wilfried G. B. Voorhorst ◽  
Yannick Gueguen ◽  
Ans C. M. Geerling ◽  
Gerti Schut ◽  
Isabell Dahlke ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Northern Blot Analysis ◽  
Pyrococcus Furiosus ◽  
Transcriptional Regulator ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Glucose Polymer ◽  
Coordinated Expression

ABSTRACT The genetic organization, expression, and regulation of thecelB locus of the hyperthermophilic archaeonPyrococcus furiosus were analyzed. This locus includes thecelB gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster,adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth ofP. furiosus on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.

scholarly journals Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c2 Gene P2 Promoter by the Response Regulator PrrA

2002 ◽  
Vol 184 (2) ◽  
pp. 390-399 ◽  
Author(s):  
James C. Comolli ◽  
Audrey J. Carl ◽  
Christine Hall ◽  
Timothy Donohue
Keyword(s):  
Amino Acid ◽  
Rhodobacter Sphaeroides ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Single Amino Acid ◽  
Single Amino Acid Change ◽  
Wide Range

ABSTRACT Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the α subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c 2 gene (cycA) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.

scholarly journals The Histone-Like Protein HU Does Not Obstruct Movement of T7 RNA Polymerase in Escherichia coli Cells but Stimulates Its Activity

2002 ◽  
Vol 184 (6) ◽  
pp. 1565-1570 ◽  
Author(s):  
Pilar Morales ◽  
Josette Rouviere-Yaniv ◽  
Marc Dreyfus
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Dna Supercoiling ◽  
Host Cells ◽  
T7 Rna Polymerase ◽  
Late Promoter ◽  
Naked Dna ◽  
Escherichia Coli Cells

ABSTRACT In vivo, RNA polymerases (RNAPs) do not transcribe naked DNA but do transcribe protein-associated DNA. Studies with the model enzyme T7 RNAP have shown that, in eukaryotic cells or in vitro, nucleosomes can inhibit both transcription initiation and elongation. We examine here whether the presence of HU, one of the major histone-like proteins in Escherichia coli cells (the genuine milieu for T7 RNAP) affects its activity. An engineered lac operon fused to the T7 late promoter was introduced into the chromosome of T7 RNAP-producing strains that either overexpress HU or lack it. The flows of RNAP that enter and exit this operon were compared with regard to the content of HU. We found that the fraction of T7 RNAP molecules that do not reach the end of the lac operon (ca. 15%) is the same whether the host cells overexpressed HU or lacked it: thus, the enzyme either freely displaces HU or transcribes through it. However, in these cells, the transcript yield was increased when HU is overexpressed and decreased in the hup mutants, presumably reflecting changes in DNA supercoiling. Thus, in contrast to eukaryotic nucleosomes, HU does not impair T7 RNAP activity but has a stimulatory effect. Finally, our results suggest that HU can also influence mRNA stability in vivo.

scholarly journals Promoters for Chlamydia Type III Secretion Genes Show a Differential Response to DNA Supercoiling That Correlates with Temporal Expression Pattern

2010 ◽  
Vol 192 (10) ◽  
pp. 2569-2574 ◽  
Author(s):  
Elizabeth Di Russo Case ◽  
Ellena M. Peterson ◽  
Ming Tan
Keyword(s):  
Rna Polymerase ◽  
Type Iii Secretion ◽  
Dna Supercoiling ◽  
Differential Response ◽  
Alternative Form ◽  
Developmental Cycle ◽  
Temporal Expression ◽  
Type Iii ◽  
Superhelical Density

ABSTRACT Type III secretion (T3S) is important for the establishment and maintenance of a chlamydial infection. The genes encoding T3S components in Chlamydia are transcribed as separate temporal classes, but the mechanisms that regulate the timing of their expression are not understood. In this study, we demonstrate that promoters for 10 predicted T3S transcriptional units are each transcribed in vitro by the major form of chlamydial RNA polymerase but not by an alternative form of RNA polymerase containing σ28. Since changes in DNA supercoiling during chlamydial development have been proposed as a mechanism for temporal gene regulation, we examined the in vitro response of T3S promoters to altered superhelical density. Promoters for three T3S genes that are upregulated at mid times were activated in response to increased DNA supercoiling. In contrast, promoters for three late T3S genes were not sensitive to changes in superhelical density. This differential response to changes in DNA topology is similar to the pattern that has been reported for representative mid and late chlamydial genes that are unrelated to the T3S system. Based on these results, we propose that the temporal expression of T3S genes in Chlamydia is controlled by general mechanisms that regulate σ66-dependent gene expression during the developmental cycle. Our results are consistent with a model in which T3S genes that are upregulated in mid cycle are activated together with other mid genes in response to increased DNA supercoiling.

High resolution mapping of nucleic acid containing complexes in vitro and in situ

1996 ◽  
Vol 54 ◽  
pp. 48-49
Author(s):  
D. P. Bazett-Jones ◽  
M. J. Hendzel
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Heavy Atom ◽  
Regulation Of Transcription ◽  
High Exposure ◽  
Resolution Mapping ◽  
Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Sign in / Sign up

Export Citation Format

Share Document