scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

1999 ◽  
Vol 19 (11) ◽  
pp. 7610-7620 ◽  
Author(s):  
Paul A. Moore ◽  
Josef Ozer ◽  
Moreh Salunek ◽  
Gwenael Jan ◽  
Dennis Zerby ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Related Protein ◽  
Multiple Promoters ◽  
Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

scholarly journals SigB-Dependent In Vitro Transcription of prfA and Some Newly Identified Genes of Listeria monocytogenes Whose Expression Is Affected by PrfA In Vivo

2005 ◽  
Vol 187 (2) ◽  
pp. 800-804 ◽  
Author(s):  
Marcus Rauch ◽  
Qin Luo ◽  
Stefanie Müller-Altrock ◽  
Werner Goebel
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Direct Link ◽  
Class Iii ◽  
New Genes ◽  
Class Iii Genes

ABSTRACT Recent studies have identified several new genes in Listeria monocytogenes which are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al., Mol. Microbiol. 47:1613-1625, 2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at the prfA promoter PprfA2 was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.

scholarly journals Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613 ◽  
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

2014 ◽  
Author(s):  
Matthew Sochor
Keyword(s):  
Transcriptional Repression ◽  
In Vitro Transcription ◽  
Lac Repressor ◽  
Molecular Crowding ◽  
Binding Data ◽  
Direct Modeling ◽  
Operator Dna ◽  
The Given

A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor-operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

scholarly journals Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

2019 ◽  
Author(s):  
Ana M. Moreno ◽  
Glaucilene F. Catroli ◽  
Fernando Alemán ◽  
Andrew Pla ◽  
Sarah A. Woller ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Genome Engineering ◽  
Therapeutic Potential ◽  
Sequence Similarity ◽  
Thermal Hyperalgesia ◽  
Loss Of Function ◽  
High Sequence Similarity ◽  
Inflammatory State

ABSTRACTCurrent treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between NaVsubtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repressionin vitroin Neuro2A cells, and then by the lumbar intrathecal route delivered both genome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of NaV1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictivepainamelioration approach enablingLong-lastingAnalgesia viaTargetedin vivoEpigeneticRepression of Nav1.7, a methodology we dubpain LATER, will have significant therapeutic potential, such as for preemptive administration in anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.One sentence summaryIn situepigenome engineering approach for genomically scarless, durable, and non-addictive management of pain.

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.

Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites

1989 ◽  
Vol 9 (9) ◽  
pp. 3603-3613
Author(s):  
J C Kennell ◽  
A M Lambowitz
Keyword(s):  
Mitochondrial Dna ◽  
Neurospora Crassa ◽  
Transcription Initiation ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription System ◽  
Group I ◽  
Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

scholarly journals Viral Hormones: Expanding Dimensions in Endocrinology

Endocrinology ◽  
2019 ◽  
Vol 160 (9) ◽  
pp. 2165-2179 ◽  
Author(s):  
Qian Huang ◽  
C Ronald Kahn ◽  
Emrah Altindis
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Endocrine System ◽  
Peptide Hormones ◽  
Rna Sequences ◽  
High Sequence Similarity ◽  
In Vivo Models ◽  
Viral Mimicry

AbstractViruses have developed different mechanisms to manipulate their hosts, including the process of viral mimicry in which viruses express important host proteins. Until recently, examples of viral mimicry were limited to mimics of growth factors and immunomodulatory proteins. Using a comprehensive bioinformatics approach, we have shown that viruses possess the DNA/RNA with potential to encode 16 different peptides with high sequence similarity to human peptide hormones and metabolically important regulatory proteins. We have characterized one of these families, the viral insulin/IGF-1–like peptides (VILPs), which we identified in four members of the Iridoviridae family. VILPs can bind to human insulin and IGF-1 receptors and stimulate classic postreceptor signaling pathways. Moreover, VILPs can stimulate glucose uptake in vitro and in vivo and stimulate DNA synthesis. DNA sequences of some VILP-carrying viruses have been identified in the human enteric virome. In addition to VILPs, sequences with homology to 15 other peptide hormones or cytokines can be identified in viral DNA/RNA sequences, some with a very high identity to hormones. Recent data by others has identified a peptide that resembles and mimics α-melanocyte-stimulating hormone’s anti-inflammatory effects in in vitro and in vivo models. Taken together, these studies reveal novel mechanisms of viral and bacterial pathogenesis in which the microbe can directly target or mimic the host endocrine system. These findings also introduce the concept of a system of microbial hormones that provides new insights into the evolution of peptide hormones, as well as potential new roles of microbial hormones in health and disease.

scholarly journals Bordetella pertussis fim3gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes

2015 ◽  
Vol 112 (6) ◽  
pp. E526-E535 ◽  
Author(s):  
Alice Boulanger ◽  
Kyung Moon ◽  
Kimberly B. Decker ◽  
Qing Chen ◽  
Leslie Knipling ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bordetella Pertussis ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Gene Activation ◽  
Virulence Gene ◽  
In Vitro Transcription ◽  
Transcription Complexes

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and theBordetella pertussisRR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the virulence genefim3(fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence offim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3activation once the signal for BvgA phosphorylation is removed.

Sign in / Sign up

Export Citation Format

Share Document