14 results on '"Strohner, Ralf"'
Search Results
2. DNA Sequence- and Conformation-Directed Positioning of Nucleosomes by Chromatin-Remodeling Complexes
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Rippe, Karsten, Schrader, Anna, Riede, Philipp, Strohner, Ralf, Lehmann, Elisabeth, and Längst, Gernot
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- 2007
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3. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties
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Tiller, Thomas, Schuster, Ingrid, Deppe, Dorothée, Siegers, Katja, Strohner, Ralf, Herrmann, Tanja, Berenguer, Marion, Poujol, Dominique, Stehle, Jennifer, Stark, Yvonne, Heling, Martin, Daubert, Daniela, Felderer, Karin, Kaden, Stefan, Kölln, Johanna, Enzelberger, Markus, and Urlinger, Stefanie
- Published
- 2013
4. The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo
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Németh, Attila, Strohner, Ralf, Grummt, Ingrid, and Längst, Gernot
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- 2004
5. Correction: High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.
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Qiu, Chenxi, Erinne, Olivia C., Dave, Jui M., Cui, Ping, Jin, Huiyan, Muthukrishnan, Nandhini, Tang, Leung K., Ganesh Babu, Sabareesh, Lam, Kenny C., Vandeventer, Paul J., Strohner, Ralf, Van den Brulle, Jan, Sze, Sing-Hoi, and Kaplan, Craig D.
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GENETIC mutation ,PHENOTYPES - Abstract
A correction to the article "High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop," by Chenxi Qiu and colleagues is presented.
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- 2018
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6. High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.
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Qiu, Chenxi, Erinne, Olivia C., Dave, Jui M., Cui, Ping, Jin, Huiyan, Muthukrishnan, Nandhini, Tang, Leung K., Babu, Sabareesh Ganesh, Lam, Kenny C., Vandeventer, Paul J., Strohner, Ralf, Van den Brulle, Jan, Sze, Sing-Hoi, and Kaplan, Craig D.
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RNA polymerases ,SACCHAROMYCES cerevisiae ,MOLECULAR biology ,FUNGAL proteins ,FUNGAL evolution ,FUNGAL gene expression - Abstract
The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins. [ABSTRACT FROM AUTHOR]
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- 2016
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7. NoRC, a novel chromatin remodeling complex involved in ribosomal RNA gene silencing
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Strohner, Ralf
- Subjects
FOS: Chemical sciences ,Chromatin remodeling, NoRC, TTF-I, rDNA transcription, gene silencing - Abstract
Regulation of gene expression takes place in the nucleus in a highly structured and condensed nucleoprotein environment, called chromatin (Felsenfeld and Groudine, 2003; Khorasanizadeh, 2004; Vaquero et al., 2003). A broad group of factors regulates the properties of chromatin; e.g. by covalently modifying histones and / or by ATP-dependent chromatin remodeling, thereby allowing or preventing gene expression. The mammalian genome contains hundreds of gene copies encoding precursor ribosomal RNA and the transcription of these genes is highly regulated with respect to cellular metabolism (Grummt, 2003). However, even in actively growing cells, only a subset of the rRNA genes are actively transcribed, exhibiting an accessible chromatin conformation (Conconi et al., 1989). In a chromatin context, the activation of rDNA genes involves the transcription termination factor TTF-I (Längst et al., 1998; Längst et al., 1997a). However, the silenced rDNA gene fraction remains in an inaccessible heterochromatic state throughout the cell cycle (Conconi et al., 1989). Until recently, the onset of silencing and the mechanisms that maintain the inactive state of rRNA genes were less understood. Recent studies, including the work presented in this thesis, provide insights into the molecular mechanism of ribosomal RNA gene silencing (Lawrence et al., 2004; Németh et al., 2004; Santoro and Grummt, 2001; Santoro et al., 2002; Strohner et al., 2004; Zhou et al., 2002). Accumulating evidence indicates that the combined action of chromatin modifying mechanisms such as chromatin remodeling, histone modification and DNA methylation contribute to the process of rRNA gene silencing. Here I present data demonstrating an active role of the chromatin remodeling complex NoRC in rDNA gene silencing and propose dual functions of TTF-I in rDNA regulation in chromatin, namely involvement in both activation and silencing of rDNA transcription. 4.1 NoRC, a novel chromatin remodeling complex In this doctoral study, a novel protein complex, composed of the nucleolar protein Tip5 and the ATPase Snf2h, was purified using convential chromatography and affinity purification methods. A detailed chromatin remodeling analysis revealed that this complex is able to induce mononucleosome movement in an ATP and histone H4 tail dependent fashion. Finally, this Tip5-Snf2h complex was termed NoRC (nucleolar remodeling complex), a novel member of the ISWI family of ATP-dependent chromatin remodeling complexes (Strohner et al., 2001). To dissect its functions, the NoRC complex was reconstituted from its recombinant subunits Tip5 and Snf2h, using the baculo virus driven expression system. Reconstitution confirmed the direct interaction between Tip5 and Snf2h. Furthermore, recombinant and cellular NoRC display similar sizes in gel filtration columns. Recombinant NoRC exhibits chromatin stimulated ATPase activity and mobilizes nucleosomes in an energy-dependent manner. Both activities are histone H4 tail dependent. NoRC and its subunits Tip5 and Snf2h were compared in different DNA / Nucleosome binding assays. NoRC shows preferred binding to structured (bent) DNA, e.g. a region within the mouse rDNA promoter, and interacts with mononucleosomes in electrophoretic mobility shift assays (EMSA). While no stable interaction with core nucleosomes could be detected in EMSA, ATPase assays and DNase I protection assays noticeably pinpointed to NoRC / nucleosome interactions with both nucleosomal and protruding linker DNA. 4.2 NoRC specifically represses rDNA transcription in chromatin The functional consequences of the Tip5 / TTF-I interaction were assessed and the influence on chromatin structure of the rDNA promoter in an in vitro system was determined. Tip5 in NoRC interacts with the N-terminal part of full length TTF-I and unmasks its DNA binding site. This interaction is required both for binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter in chromatin. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome. To elucidate a potential role of NoRC in rDNA transcriptional regulation, we used an in vitro transcription system with an rDNA minigene reconstituted into chromatin. These studies revealed a specific function for NoRC in rDNA transcriptional repression on chromatin templates. In contrast, NoRC had no effect on DNA transcription. Transcription experiments were then performed with chromatin templates reconstituted from recombinant histones lacking individual histone tails. The results indicate that NoRC-mediated rDNA gene repression is dependent on the histone H4 tail, suggesting an involvement of chromatin remodeling. Further transcription experiments revealed that NoRC-mediated repression occurs prior to preinitiation complex formation and does not affect activated rDNA genes. NoRC stably associates with the silenced gene, and these early steps of rDNA repression do not depend on DNA and histone modifications (Strohner et al., 2004). NoRC showed preferred binding to a structured (bent) region within the mouse rDNA promoter. Methylation of a single CpG dinucleotide within this region abrogated rDNA transcription in chromatin (Santoro and Grummt, 2001), but did not influence DNA binding of NoRC. Furthermore, nucleosomal DNA is less methylated than free DNA, but chromatin remodeling enhances methylation. The results suggest an important role for the chromatin remodeling complex NoRC in the establishment of rDNA silencing. NoRC then contributes to maintenance of the silenced state throughout the cell cycle by interacting with DNA and histone modifying enzymes. Transcriptional repression by chromatin remodeling factors seems to be a common mechanism to stably inhibit gene expression.
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- 2004
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8. Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody.
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Eylenstein, Roy, Weinfurtner, Daniel, Härtle, Stefan, Strohner, Ralf, Böttcher, Jark, Augustin, Martin, Ostendorp, Ralf, and Steidl, Stefan
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- 2016
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9. Chromatin Targeting Signals, Nucleosome Positioning Mechanism and Non-Coding RNA-Mediated Regulation of the Chromatin Remodeling Complex NoRC.
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Manelyte, Laura, Strohner, Ralf, Gross, Thomas, and Längst, Gernot
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CHROMATIN , *RIBOSOMAL RNA , *EPIGENETICS , *NUCLEOPROTEINS , *CHROMOSOMES - Abstract
Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC. [ABSTRACT FROM AUTHOR]
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- 2014
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10. A 'loop recapture' mechanism for ACF-dependent nucleosome remodeling.
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Strohner, Ralf, Wachsmuth, Malte, Dachauer, Karoline, Mazurkiewicz, Jacek, Hochstatter, Julia, Rippe, Karsten, and Längst, Gernot
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CHROMATIN , *DNA , *ADENOSINE triphosphatase , *CHROMOSOMES , *NUCLEOPROTEINS , *MOLECULAR biology - Abstract
The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning. [ABSTRACT FROM AUTHOR]
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- 2005
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11. Recruitment of the Nucleolar Remodeling Complex NoRC Establishes Ribosomal DNA Silencing in Chromatin.
- Author
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Strohner, Ralf, Németh, Attila, Nightingale, Karl P., Grummt, Ingrid, Becker, Peter B., and Längst, Gernot
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CHROMATIN , *RIBOSOMES , *DNA , *DNA polymerases , *CYTOLOGY , *MOLECULAR biology - Abstract
The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TFF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing. [ABSTRACT FROM AUTHOR]
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- 2004
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12. Maximizing Detergent Stability and Functional Expression of a GPCR by Exhaustive Recombination and Evolution
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Schlinkmann, Karola M., Hillenbrand, Matthias, Rittner, Alexander, Künz, Madeleine, Strohner, Ralf, and Plückthun, Andreas
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G protein coupled receptors , *GENE expression , *BIOSYNTHESIS , *BIOLOGICAL membranes , *RECOMBINANT DNA , *AMINO acids , *ETHYLENEDIAMINETETRAACETIC acid - Abstract
Abstract: To identify structural features in a G‐protein‐coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several “shift” positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G‐protein. We discuss the structural reasons for these improvements achieved through directed evolution. [Copyright &y& Elsevier]
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- 2012
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13. Synthetic Antibodies Designed on Natural Sequence Landscapes
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Zhai, Wenwu, Glanville, Jacob, Fuhrmann, Markus, Mei, Li, Ni, Irene, Sundar, Purnima D., Van Blarcom, Thomas, Abdiche, Yasmina, Lindquist, Kevin, Strohner, Ralf, Telman, Dilduz, Cappuccilli, Guido, Finlay, William J.J., Van den Brulle, Jan, Cox, David R., Pons, Jaume, and Rajpal, Arvind
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IMMUNOGLOBULINS , *AMINO acid sequence , *CLUSTERING of particles , *GENE expression , *GENE libraries , *CELL receptors , *PEROXIDASE , *IMMUNOGENETICS - Abstract
Abstract: We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (VH) and two kappa (Vκ) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all VH and Vκ sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×1010 transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×105 clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy. [Copyright &y& Elsevier]
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- 2011
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14. The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding.
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Bonaldi T, Längst G, Strohner R, Becker PB, and Bianchi ME
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- Animals, Binding Sites, DNA Footprinting, Dose-Response Relationship, Drug, Drosophila, Mice, Models, Biological, Protein Binding, Time Factors, Chromatin metabolism, DNA metabolism, HMGB1 Protein metabolism, Mutation, Nucleosomes metabolism, Recombinant Proteins metabolism
- Abstract
Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling.
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- 2002
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