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Single-Pair Fluorescence Resonance Energy Transfer (FRET) experiments reveal structural and dynamic information about macro-molecules by monitoring the change in FRET efficiency between fluorescent dyes attached to a macromolecule. The Nano-Positioning System (NPS) developed recently  uses data from several of such experiments to infer the position of a dye attached to protein sites unresolved by x-ray crystallography. Briefly, we perform probabilistic data analysis that allows us todoi:10.1016/j.bpj.2009.12.3189 fatcat:wvm6gjvrgfb3bd4ieudmexi5ia
more »... e the distribution of possible dye positions in a simple and objective way without relying on ad-hoc procedures. Up to now NPS was limited to the triangulation of just one fluorescently labelled position based on FRET measurements to several other positions known from crystal structure [1,2]. Here, we discuss ways to extend the present model beyond this basic triangulation principle. In particular, we show how to gain three dimensional distance information by analysing triangulation networks where FRET is measured between arbitrary labelling sites in absence of other structural information.  A.
1 , Josef Exler 2 , Gernot Längst 2 , Jens Michaelis 1,3 . 1 Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität (LMU), München, Germany, 2 Department of Biochemistry III, University ... RNAs induced by protein binding with minimal interference from protein aggregation. 1485-Pos Board B329 Single-pair Fluorescence Resonance Energy Transfer study of mononucleosomes dynamics Barbara Treutlein ...doi:10.1016/j.bpj.2008.12.1447 fatcat:vbgn4yxijnd5ng62nrrvolx7uy
Michaelis 1 , Wolfgang Kügel 1 , Barbara Treutlein 1 , Josef Exler 2 , Gernot ängst 2 . 1 Ludwigs Maximilians University, Munich, Germany, 2 University of Regensburg, Regensburg, Germany. ... is very suitable to extend to other remodelers exhibiting different modes of action. 2457-Pos 2458-Pos ATP Dependent Nucleosome Remodelling -Mechanistic Insights from Single Molecule Experiments Jens ...doi:10.1016/j.bpj.2009.12.2583 fatcat:jmgvnyfyhnfp5nb44m2bfsdfwm
, Jens Michaelis. ... Treutlein, M.A. Izquierdo Arcusa, A. Muschielok, R. Lewis; A.C.M. Cheung, P. Cramer, and J. Michaelis, NAR doi:10.1093/nar/ gkp601 (2009). ...doi:10.1016/j.bpj.2009.12.1120 fatcat:2cojip5vcrghdjafma4lw2gh7q
, Adam Muschielok, Daniel Ryan, Tom Owen-Hughes, Jens Michaelis. ... , Adam Muschielok, Joanna Andrecka, Anass Jawhari, Claudia Buchen, Friederike Hög, Patrick Cramer, Jens Michaelis. ...doi:10.1016/j.bpj.2010.12.2786 fatcat:hd3vcdssffb5lcnb5p2ehe72pu
High quality biomaterial from patients and controls is a core prerequisite for research into the biological causes of mental disorder. BioPsy is the biobank of the Department of Genetic Epidemiology in Psychiatry at the Central Institute of Mental Health in Mannheim, Germany, and one of the largest psychiatric disorder biobanks worldwide. Here, ongoing collection is in progress for blood, DNA, mRNA, plasma, serum, saliva, urine, hair, and other biomaterials. Reuse of samples is permitted in adoi:10.5334/ojb.18 fatcat:kkprcvlvlbefdammcd2ouz3z7m
more »... llaboration-based context. BioPsy operates according to German and European quality and data privacy standards.
Reward system dysfunction is implicated in the pathogenesis of major psychiatric disorders. We conducted a genome-wide association study (GWAS) to identify genes that influence activation strength of brain regions within the extended reward system in humans. A large homogeneous sample of 214 participants was genotyped and underwent functional magnetic resonance imaging (fMRI). All subjects performed the desire-reason dilemma (DRD) paradigm allowing systematic investigation of systems-leveldoi:10.1101/2022.05.03.490395 fatcat:i2cahfxkxbe2fe3a7cjwdgptti
more »... nisms of reward processing in humans. As a main finding, we identified the single nucleotide variant rs113408797 in the DnaJ Heat Shock Protein Family Member C13 gene (DNAJC13, alias RME-8), that strongly influenced the activation of the ventral tegmental area (VTA; p = 2.50E-07) and the nucleus accumbens (NAcc; p = 5.31E-05) in response to conditioned reward stimuli. Moreover, haplotype analysis assessing the information across the entire DNAJC13 locus demonstrated an impact of a five-marker haplotype on VTA activation (p = 3.21E-07), which further corroborates a link between this gene and reward processing. The present findings provide first direct empirical evidence that genetic variation of DNAJC13 influences neural responses within the extended reward system to conditioned stimuli. Further studies are required to investigate the role of this gene in the pathogenesis and pathophysiology of neuropsychiatric disorders.
Transcription initiation of eukaryotic protein-coding genes is a multi-step process that requires assembly of RNA polymerase (Pol) II and the general transcription factors into the closed complex. Then, the DNA surrounding the TSS is melted and inserted into the Pol II active center cleft to form the open complex (OC), the starting point for RNA synthesis. The OC is hence a central intermediate in the initiation-elongation transition and insights into its structure are crucial for understandingdoi:10.1016/j.bpj.2011.11.1574 fatcat:dwmtxeto2nak3fw6dtwu5ezjli
more »... the molecular mechanism of transcription initiation. X-ray crystallography has provided detailed insights into the architecture of Pol II in different functional states, but structural studies of transcription initiation have been hindered by the flexibility of Pol II initiation complexes. As a consequence, the structure of the OC remains unknown. Here, we employ single-molecule Förster Resonance Energy Transfer (smFRET) and Nano-Positioning System (NPS)analysis  to determine the 3-dimensional architecture of a minimal OC consisting of Pol II, promoter DNA, TBP, TFIIB, and -IIF. Briefly, by using smFRET, we measure distances within double labeled OCs and the NPS computes 3-dimensional position probability densities of unknown sites on upstream DNA, TATA-DNA, TBP, and TFIIB, which allows us to build a model of the OC. We show that in the OC, TBP and TATA-DNA reside above the Pol II cleft between clamp and protrusion. Downstream DNA is dynamically loaded into the cleft and unloaded from the cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it was located in the closed complex. Hence, our results define the overall structural changes during the initiation-elongation transition, and reveal a key role for the intrinsic flexibility of TFIIB in accommodating these changes.  Muschielok et al., Nature Methods, 5, 965 (2008). A long-standing key question in the transcription field has been how Pol II achieves such a high transcriptional fidelity, with an error rate ranging between 10 À4 and 10 À5 . Maintaining transcriptional fidelity is vital to cells and organisms. Novel insights into this subject have recently emerged from a combination of structural, genetic and biochemical studies, suggesting that the high accuracy of Pol II transcription is achieved by specific nucleotide selection and by removal of misincorporated nucleotides (proofreading). Here, we use a combined approach including pre-steady state transient kinetics, X-ray crystallography, and molecular dynamics simulations to elucidate the roles of electrostatic effects, such as hydrogen bonds, and of steric effects in controlling Pol II transcription fidelity. RNA polymerase II (RNAPII) carries out the transcription of all mRNA in eukaryotic cells. During transcript elongation, RNAPII must select the correct nucleotide, catalyze its addition to the growing RNA chain, and move stepwise along the template DNA until the gene is fully transcribed. The trigger loop is an evolutionarily conserved protein subdomain that has been implicated in substrate selection and catalysis in multi-subunit RNA polymerases, but its role in the elongation process is not fully elucidated. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motions of individual wild-type and trigger-loop mutant RNAPII mole-cules. We report direct evidence for trigger loop involvement in the RNAPII translocation event. Global fits to the force-velocity relationships for RNAPII and its trigger loop mutants support a Brownian-ratchet model for elongation, where the incoming NTP is able to bind in either pre-or post-translocated states, and movement between these two states is governed by the trigger loop. Under conditions that promote misincorporation, the observed pausing kinetics suggest that the trigger loop governs fidelity in both substrate selection and mismatch recognition. RNA polymerases are characterized by conflicting requirements during initial transcription and elongation. In the initial phase, an RNA polymerase must recognize a specific promoter sequence, melt the DNA at the start site, initiate de novo synthesis of a dimer RNA, and extend that RNA in a hybrid that is thermodynamically unstable to collapse of the melted bubble. Hybrid growth in turn ultimately drives promoter release and a structural transition that enables topologically locked RNA entry into a stabilizing exit channel, establishing a stable elongation complex. Perhaps not surprisingly, short RNAs are not stable during initial transcription, leading to their abortive release. But hybrid stability is only part of the story. In T7 RNA polymerase, a single point mutation (P266L) in the protein, distant from the hybrid, leads to a dramatic reduction in abortive release on most promoters. Current models suggest that the mutant releases promoter contacts more readily, transitioning to a stable elongation complex sooner. New results reveal the opposite behavior: P266L releases promoter contacts later than wild type, suggesting an overall increase in stability of the initially transcribing complex. Based on kinetic studies, we present a testable model to explain how growth of the hybrid serves to drive a timed transition to elongation. Transcription of DNA into RNA is the first step in gene expression and is therefore a common target of regulation. The last few decades have witnessed a significant advance in our understanding of the biochemical mechanisms underlying RNA synthesis by RNA polymerase (RNAP). Despite many phenomenological observations establishing a functional relationship between DNA template mechanics and transcription, very little is known about how the mechanical features of DNA interact with RNA polymerase on a mechanistic level. To shed light on how DNA bending and torsional stresses impact RNAP function, we examine the mechanical interaction of bacteriophage T7-RNAP with several circular DNA fragments that differ in length and linking number. In particular, we employ molecular dynamics to simulate the interaction of T7-RNAP with the following three DNA minicircles: 100 base pair(bp) with linking number 9 (Lk=9), 106-bp with Lk=10, and 108-bp with Lk=10. Each of these corresponds respectively to underwound, overwound, and relaxed configurations. The simulations reveal that torque from T7RNAP induces local relaxation of the bend angle of the circular DNA motif. Furthermore, T7RNAP induces out-of-plane deformation of highly strained DNA minicircles. Different torsional stresses seem to impact DNA conformation to various degrees: change in internal torque (undertwisting/overtwisting DNA) forces circular DNA complexed with T7RNAP to contort into a new shape. Based on these observations, we investigate the following: i) how the shape of a supercoiled DNA molecule impacts the barrier for the polymerase translocation step and ii) why DNA twist and bend angles are important factors in determining the transcription rate on strained templates.
A functional polymorphism (val158met) of the gene coding for Catechol-O-methyltransferase (COM) has been demonstrated to be related to processing of emotional stimuli. Also, this polymorphism has been found to be associated with pain regulation in healthy subjects. Therefore, we investigated a possible influence of this polymorphism on pain processing in healthy persons as well as in subjects with markedly reduced pain sensitivity in the context of Borderline Personality Disorder (BPD). Fiftydoi:10.1371/journal.pone.0023658 pmid:22247753 pmcid:PMC3256133 fatcat:ozht4vb2wfgixhe4uiqoer2ihq
more »... males (25 patients with BPD and 25 healthy control participants) were included in this study. Genotype had a significant -though moderate -effect on pain sensitivity, but only in healthies. The number of val alleles was correlated with the BOLD response in several pain-processing brain regions, including dorsolateral prefrontal cortex, posterior parietal cortex, lateral globus pallidus, anterior and posterior insula. Within the subgroup of healthy participants, the number of val alleles was positively correlated with the BOLD response in posterior parietal, posterior cingulate, and dorsolateral prefrontal cortex. BPD patients revealed a positive correlation between the number of val alleles and BOLD signal in anterior and posterior insula. Thus, our data show that the val158met polymorphism in the COMT gene contributes significantly to inter-individual differences in neural pain processing: in healthy people, this polymorphism was more related to cognitive aspects of pain processing, whereas BPD patients with reduced pain sensitivity showed an association with activity in brain regions related to affective pain processing.
The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ singlemolecule Fö rster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and generaldoi:10.1016/j.molcel.2012.02.008 pmid:22424775 fatcat:jgft55qr6janjbil3aiddigxaq
more »... ion factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.
For rbcL data we derived a calibration of 1% sequence divergence equalling 28 million years (Treutlein, 1998) . ...doi:10.1016/s0305-1978(00)00064-8 pmid:11152945 fatcat:cnktz6hnrfac7dy3nv6i7uu6ze
, Adam Muschielok, Daniel Ryan, Tom Owen-Hughes, Jens Michaelis. ... , Adam Muschielok, Joanna Andrecka, Anass Jawhari, Claudia Buchen, Friederike Hög, Patrick Cramer, Jens Michaelis. ...doi:10.1016/j.bpj.2010.12.2787 fatcat:crponfw5czb45mjotao37wovc4
Author's response to reviews: see over BMC Psychiatry Editorial Office Sabina Alam, PhD Re-Submission of manuscript Dear Dr. Alam, First, we would like to thank the referees for their comments. Their criticism gave us the opportunity to clarify our thoughts and to improve the manuscript. In the following, we will respond explicitly to their remarks. Reviewer 1 Th first reviewer describes the article as "an article of importance in its field". We thank the reviewer for his comment. Reviewer 2doi:10.1186/1471-244x-9-60 pmid:19778423 pmcid:PMC2761379 fatcat:kjhthevtjnajppjxfvixscw6zi
more »... second reviewer criticizes three major points: Comment 1: The scholarship was not first rate. Several newer, relevant studies, including those linking G72 expression to the mitochondria and an Opgen-Rhein study examining G72 in relation to cognition, were not included. In the revised verion of the manuscript, we describe the functional mechanism of G72 now in more detail. We also included newer studies linking G72 expression to the mitochondria. The revised version of our manuscript (page 10) now reads as . Thus, G72 might work as an indirect modulator of NMDA neurotransmission, which has been implicated in various cognitive domains. Lower serum level of D-serine has been shown, for instance, in patients with schizophrenia. Furthermore, the administration of D-serine (as add-on medication) has been shown to reduce some of the symptoms in schizophrenia . This provides a potential link between G72 and the glutamate hypofunction hypothesis of schizophrenia . Another study however failed to confirm the interaction between G72 and DAAO . Rather, LG72, a splicing isoform of the G72 gene, encodes for a mitrochondrial protein. It was shown that an overexpression of G72 led to mitrochondrial fragmentation. The authors proposed that an unknown function of the G72 in modulating mitochondrial morphology might be responsible for the risk-conferring property of the gene." "The functional mechanisms of G72 are still not fully understood. Chumakov and colleagues showed that the G72 protein (which is only known in higher primates) activates a second protein, D-amino acid oxidase (DAAO), that is involved in the mechanisms of D-serine . D-serine is an agonist at the glycine modulation site of the N-methyl-D-aspartate (NMDA) receptor
Single-molecule biophysics experiments have been widely employed to characterize the mechanics and dynamics of DNA and the activity of DNA-binding proteins. In typical experiments requiring DNA tethering, a DNA molecule is conjugated to a coverslip and observed with a light microscope parallel to the optical axis, limiting the resolution of optical and magnetic trapping measurements and direct observation of interactions with DNA-binding proteins. Another assay technique, where two beadsdoi:10.1016/j.bpj.2008.12.1439 fatcat:3zp4ru7zpzexfcgqx4qg7mrshm
more »... ng DNA are optically trapped, orients the DNA perpendicular to the optical axis, but this assay is limited to manipulating one DNA molecule at a time. We developed a device to overcome the limitations of previous assays. A microfabricated cantilever platform exposes the edge of a 50 nm thin gold surface that runs along the length of the sidewalls of patterned microfluidic channels. This narrow gold surface, oriented orthogonal to the optical axis, serves as a substrate for the covalent attachment of an end-thiolated DNA molecule through well established goldthiol chemistry. The microfabricated cantilever device provides complete optical access to the DNA, and allows a DNA-tethered bead to be optically or magnetically trapped perpendicular to the optical axis, the experimentally preferred orientation. This experimental set-up allows for efficient, parallelized investi- 1477-Pos Board B321 Position of the non-template DNA in the Polymerase II elongation complex revealed using single-molecule Fluorescence Resonance Energy Transfer
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