Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules.

Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences.

The XXmotif web server for eXhaustive, weight matriX-based motif discovery in nucleotide sequences.

The discovery of regulatory motifs enriched in sets of DNA or RNA sequences is fundamental to the analysis of a great variety of functional genomics experiments. These motifs usually represent binding sites of proteins or non-coding RNAs, which are best described by position weight matrices (PWMs). We have recently developed XXmotif, a de novo motif discovery method that is able to directly optimize the statistical significance of PWMs.

Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins.

Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However, to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput method to study biochemical and functional characteristics of fluorescent fusion proteins.

Downregulation of the Hsp90 system causes defects in muscle cells of Caenorhabditis elegans.

The ATP-dependent molecular chaperone Hsp90 is required for the activation of a variety of client proteins involved in various cellular processes. Despite the abundance of known client proteins, functions of Hsp90 in the organismal context are not fully explored. In Caenorhabditis elegans, Hsp90 (DAF-21) has been implicated in the regulation of the stress-resistant dauer state, in chemosensing and in gonad formation.

A fluorescence polarization assay for the discovery of inhibitors of the polo-box domain of polo-like kinase 1.

Polo-like kinase 1 (Plk1) is a key player in mitosis and has been widely recognized as a therapeutic target for many human cancer types. Apart from its kinase domain, Plk1 harbors a protein-protein interaction domain dubbed "polo-box domain" (PBD), by which the enzyme binds to its intracellular anchorage sites and to at least a fraction of its substrates. Recent evidence indicates that the inhibition of the PBD by small molecules is feasible and might allow for the discovery of highly specific inhibitors of the enzyme.

Transcription modulation chromosome-wide: universal features and principles of dosage compensation in worms and flies.

Dosage compensation processes in flies and worms provide a unique opportunity to study common regulatory principles of thousands of genes. Technological advancement in the recent years has allowed for the comprehensive description of key aspects such as the targeting of the regulatory factors, the emerging chromatin structure changes and the ensuing subtle transcriptional alterations. With plenty of data at hand the challenge remains to integrate the findings into coherent models that appreciate the global nature of the underlying principles leaving the experimental anecdotes behind while avoiding the numerical burlesque.

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region.

Glial cell line-derived neurotrophic factor is constitutively produced by human testicular peritubular cells and may contribute to the spermatogonial stem cell niche in man.

Background: Testicular peritubular cells form an ill-characterized cellular compartment of the human testis, which forms a border with Sertoli cells and spermatogonial stem cells (SSCs). A recently developed culture method has identified parts of the secretory repertoire of human testicular peritubular cells (HTPCs), which includes nerve growth factor. Whether peritubular cells produce glial cell line-derived neurotrophic factor (GDNF) and may thus contribute to the stem cell niche is not known.

Increasing the efficiency of the ring-opening reaction of photochromic indolylfulgides by optical pre-excitation.

For three indolylfulgides the quantum efficiency of the ring-opening reaction upon pre-excitation is investigated in a multipulse experiment. The quantum efficiency grows by factor of up to 3.4, when the pre-excitation pulse immediately precedes the excitation process.

Stability and reaction dynamics of trifluorinated indolylfulgides.

Quantum efficiencies and ultrafast dynamics of the ring-closure and ring-opening reaction of a trifluorinated dicyclopropyl indolylfulgide with improved photostability are investigated by stationary and ultrafast absorption spectroscopy. The ring-closure reaction occurs on the time scale of 200 fs and is found to be temperature independent (T = 287 - 333 K). However, an activated behaviour is observed for the ring-opening reaction.

The impact of amino acid side chain mutations in conformational design of peptides and proteins.

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry.

Structural insight into nascent polypeptide chain-mediated translational stalling.

Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom-resolution cryo-electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene.

RNA polymerase II-TFIIB structure and mechanism of transcription initiation.

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II-B complex at 4.3 A resolution, and complementary functional data.

Molecular basis of transcriptional mutagenesis at 8-oxoguanine.

Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site.

A movie of the RNA polymerase nucleotide addition cycle.

During gene transcription, RNA polymerase (Pol) passes through repetitive cycles of adding a nucleotide to the growing mRNA chain. Here we obtained a movie of the nucleotide addition cycle by combining structural information on different functional states of the Pol II elongation complex (EC). The movie illustrates the two-step loading of the nucleoside triphosphate (NTP) substrate, closure of the active site for catalytic nucleotide incorporation, and the presumed two-step translocation of DNA and RNA, which is accompanied by coordinated conformational changes in the polymerase bridge helix and trigger loop.

Identification, structure, and functional requirement of the Mediator submodule Med7N/31.

Mediator is a modular multiprotein complex required for regulated transcription by RNA polymerase (Pol) II. Here, we show that the middle module of the Mediator core contains a submodule of unique structure and function that comprises the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1). The Med7N/31 submodule shows a conserved novel fold, with two proline-rich stretches in Med7N wrapping around the right-handed four-helix bundle of Med31.

Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex.

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical.

Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation.

To study how RNA polymerase II translocates after nucleotide incorporation, we prepared elongation complex crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the elongation complex in a new state, which was refined at 3.4-A resolution and identified as a possible translocation intermediate.