Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor

1989 ◽  
Vol 9 (11) ◽  
pp. 5123-5133
Author(s):  
E H Postel ◽  
S E Mango ◽  
S J Flint
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Factor ◽  
Mobility Shift ◽  
Cis Acting ◽  
Factor Binding ◽  
Myc Oncogene ◽  
Transcription In Vitro

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

scholarly journals A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.

1989 ◽  
Vol 9 (11) ◽  
pp. 5123-5133 ◽  
Author(s):  
E H Postel ◽  
S E Mango ◽  
S J Flint
Keyword(s):  
Transcription Factor ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Factor ◽  
Mobility Shift ◽  
Cis Acting ◽  
Factor Binding ◽  
Myc Oncogene ◽  
Transcription In Vitro

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

Regulation of entry into gametogenesis by Ste11: the endless game

2013 ◽  
Vol 41 (6) ◽  
pp. 1673-1678 ◽  
Author(s):  
Jayamani Anandhakumar ◽  
Sylvain Fauquenoy ◽  
Philippe Materne ◽  
Valérie Migeot ◽  
Damien Hermand
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Molecular Mechanisms ◽  
Fundamental Aspect ◽  
Master Regulator ◽  
Proliferation And Differentiation ◽  
Fate Decision ◽  
High Level ◽  
Transcriptional Controls

Sexual reproduction is a fundamental aspect of eukaryotic cells, and a conserved feature of gametogenesis is its dependency on a master regulator. The ste11 gene was isolated more than 20 years ago by the Yamamoto laboratory as a suppressor of the uncontrolled meiosis driven by a pat1 mutant. Numerous studies from this laboratory and others have established the role of the Ste11 transcription factor as the master regulator of the switch between proliferation and differentiation in fission yeast. The transcriptional and post-transcriptional controls of ste11 expression are intricate, but most are not redundant. Whereas the transcriptional controls ensure that the gene is transcribed at a high level only when nutrients are rare, the post-transcriptional controls restrict the ability of Ste11 to function as a transcription factor to the G1-phase of the cell cycle from where the differentiation programme is initiated. Several feedback loops ensure that the cell fate decision is irreversible. The complete panel of molecular mechanisms operating to warrant the timely expression of the ste11 gene and its encoded protein basically mirrors the advances in the understanding of the numerous ways by which gene expression can be modulated.

Electron spectroscopic imaging of encapsidated DNA in vaccinia virus

1995 ◽  
Vol 41 (10) ◽  
pp. 889-894 ◽  
Author(s):  
G. Harauz ◽  
D. H. Evans ◽  
D. R. Beniac ◽  
A. L. Arsenault ◽  
B. Rutherford ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Central Element ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
The Core ◽  
Dna Backbone ◽  
Ultrathin Sections

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.Key words: vaccinia, electron spectroscopic imaging, DNA.

scholarly journals Mutational Analysis of Escherichia coli Heat Shock Transcription Factor Sigma 32 Reveals Similarities with Sigma 70 in Recognition of the −35 Promoter Element and Differences in Promoter DNA Melting and −10 Recognition

2005 ◽  
Vol 187 (19) ◽  
pp. 6762-6769 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Laura Tsujikawa ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Amino Acid ◽  
Heat Shock ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Promoter Dna

ABSTRACT Upon the exposure of Escherichia coli to high temperature (heat shock), cellular levels of the transcription factor σ32 rise greatly, resulting in the increased formation of the σ32 holoenzyme, which is capable of transcription initiation at heat shock promoters. Higher levels of heat shock proteins render the cell better able to cope with the effects of higher temperatures. To conduct structure-function studies on σ32 in vivo, we have carried out site-directed mutagenesis and employed a previously developed system involving σ32 expression from one plasmid and a β-galactosidase reporter gene driven by the σ32-dependent groE promoter on another in order to monitor the effects of single amino acid substitutions on σ32 activity. It was found that the recognition of the −35 region involves similar amino acid residues in regions 4.2 of E. coli σ32 and σ70. Three conserved amino acids in region 2.3 of σ32 were found to be only marginally important in determining activity in vivo. Differences between σ32 and σ70 in the effects of mutation in region 2.4 on the activities of the two sigma factors are consistent with the pronounced differences between both the amino acid sequences in this region and the recognized promoter DNA sequences.

scholarly journals Transcriptional Control of the Sulfur-RegulatedcysH Operon, Containing Genes Involved inl-Cysteine Biosynthesis in Bacillus subtilis

2000 ◽  
Vol 182 (20) ◽  
pp. 5885-5892 ◽  
Author(s):  
Maria Cecilia Mansilla ◽  
Daniela Albanesi ◽  
Diego de Mendoza
Keyword(s):  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Sulfur Metabolism ◽  
Transcriptional Level ◽  
Cysteine Biosynthesis ◽  
Gram Positive ◽  
Sulfur Starvation ◽  
High Level

ABSTRACT The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysHoperon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH, cysP,ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. ThecysH operon contains a 5′ leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the −35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O-acetyl-l-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysHoperon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.

Electron Spectroscopic Imaging of DNA and Protein-DNA Complexes

1988 ◽  
Vol 46 ◽  
pp. 172-173
Author(s):  
D.P. Bazett-Jones ◽  
M.L. Brown
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Electron Microscope ◽  
Spatial Resolution ◽  
Spectroscopic Imaging ◽  
X Rays ◽  
Electron Spectrometer ◽  
Electron Spectroscopic Imaging ◽  
Dna Complexes ◽  
Fixed Beam

Elemental distributions in cells and molecular spreads can now be produced at the spatial resolution attainable in the electron microscope by the collection of X-rays or by the collection of and imaging with inellastically scattered electrons. With the latter method, known as Electron Spectroscopic Imaging (ESI), an image is produced with electrons that have lost characteristic amounts of energy from ionizing or exciting specific elements in the specimen. ESI can generate an elemental map of a specimen at a resolution of about 0.5 nm. It can be carried out in a fixed beam microscope equipped with a parallel energy filter inserted into the column of the microscope below the specimen (1,2). An instrument equipped with a prism-mirror-prism electron spectrometer was used in this study to image purified DNA molecules and a complex of the transcription factor TFIIIA with DNA.Transcription of most genes is activated by the binding of transcription factors to promoter elements.

Transcription of the chicken Grin1 gene is regulated by the activity of SP3 and NRSF in undifferentiated cells and neurons

2008 ◽  
Vol 28 (4) ◽  
pp. 177-188 ◽  
Author(s):  
Gabriel Moreno-González ◽  
Ana María López-Colomé ◽  
Gabriela Rodríguez ◽  
Angel Zarain-Herzberg
Keyword(s):  
Transcription Factor ◽  
Gene Transcription ◽  
Cortical Neurons ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Regulatory Region ◽  
Initiation Site ◽  
Undifferentiated Cells ◽  
The Central Nervous System

The NMDA (N-methyl-D-aspartate) receptors are important in the regulation of neuronal development, synaptic plasticity, learning and memory, and are involved in several brain pathologies. The NR1 subunit is essential for the assembly of functional receptors, as it forms the calcium-permeable ion channel and contains the obligatory co-agonist binding site. Previous studies have shown that NR1 gene (Grin1) expression is up-regulated during neuronal differentiation and its expression is widespread in the central nervous system. We have previously cloned the chicken Grin1 gene and 1.9 kb of the 5′-regulatory region. In the present study, we analysed the molecular mechanisms that regulate chicken Grin1 gene transcription in undifferentiated cells and neurons. By functional analysis of chicken Grin1–luciferase gene 5′-regulatory region constructs, we demonstrate that the basal promoter is delimited within 210 bp upstream from the main transcription initiation site. DNA–protein binding and functional assays revealed that the 5′-UTR (untranslated region) has one consensus NRSE (neuron-restrictive silencing element) that binds NRSF (neuron-restrictive silencing factor), and one SP (stimulating protein transcription factor) element that binds SP3, both repressing Grin1 gene transcription in undifferentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells (phaeochromocytoma cells). The promoter region lacks a consensus TATA box, but contains one GSG/SP (GSG-like box near a SP-consensus site) that binds SP3 and up-regulates gene transcription in embryonic chicken cortical neurons. Taken together, these results demonstrate a dual role of SP3 in regulating the expression of the Grin1 gene, by repressing transcription in the 5′-UTR in undifferentiated cells as well as acting as a transcription factor, increasing Grin1 gene transcription in neurons.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

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