Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of ...the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30–35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase “clamp head loop” and the TFIIF “charged region” that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.
Display omitted
•High-resolution structure of yeast PIC fully resolves transcription factors IIE and IIH•PIC intermediate provides insights into initial DNA opening in Pol II initiation•Pol II clamp head loop and TFIIF charged region stabilize the initial 6 nt DNA bubble•Sensitive in vitro transcription assay assigns roles to regulatory PIC elements
Schilbach et al. provide a high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening in transcription.
The transition from transcription initiation to elongation is highly regulated in human cells but remains incompletely understood at the structural level. In particular, it is unclear how ...interactions between RNA polymerase II (RNA Pol II) and initiation factors are broken to enable promoter escape. Here, we reconstitute RNA Pol II promoter escape in vitro and determine high-resolution structures of initially transcribing complexes containing 8-, 10-, and 12-nt ordered RNAs and two elongation complexes containing 14-nt RNAs. We suggest that promoter escape occurs in three major steps. First, the growing RNA displaces the B-reader element of the initiation factor TFIIB without evicting TFIIB. Second, the rewinding of the transcription bubble coincides with the eviction of TFIIA, TFIIB, and TBP. Third, the binding of DSIF and NELF facilitates TFIIE and TFIIH dissociation, establishing the paused elongation complex. This three-step model for promoter escape fills a gap in our understanding of the initiation-elongation transition of RNA Pol II transcription.
Display omitted
•Visualization of initiation-elongation transition intermediates de novo initiated from the PIC•Initial RNA synthesis repositions TFIIB reader but does not displace TFIIB•Transcription bubble rewinding coincides with the displacement of TFIIA, TBP, and TFIIB•Recruitment of DSIF and NELF facilitates the displacement of TFIIE and TFIIH
Zhan et al. reconstitute the initiation-elongation transition of RNA polymerase II (RNA Pol II) and provide high-resolution structures of transcription intermediates during promoter escape. They conclude on the molecular mechanisms of promoter escape by mammalian Pol II.
At active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further ...upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal +1 nucleosome can reduce RNA synthesis in vivo and in vitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the +1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10 bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation.
Display omitted
•Promoter proximity of the +1 nucleosome reduces transcription in vivo and in vitro•The +1 nucleosome impairs transcription by interfering with productive PIC assembly•TFIIH can bind to the PIC in its closed conformation, but in an inhibited state•Transcription regulation by the +1 nucleosome depends on its proximity to the TSS
Through functional assays and cryoelectron microscopy, Abril-Garrido et al. elucidated a mechanism by which the +1 nucleosome reduces transcription. When the +1 nucleosome is located in proximity to the gene promoter, the PIC assembles—albeit in an inhibited state incompatible with DNA opening.
The approximately 350 ion channels encoded by the mammalian genome are a main pillar of the nervous system. We have determined the expression pattern of 320 channels in the two-week-old (P14) rat ...brain by means of non-radioactive robotic in situ hybridization. Optimized methods were developed and implemented to generate stringently coronal brain sections. The use of standardized methods permits a direct comparison of expression patterns across the entire ion channel expression pattern data set and facilitates recognizing ion channel co-expression. All expression data are made publically available at the Genepaint.org database. Inwardly rectifying potassium channels (Kir, encoded by the
Kcnj
genes) regulate a broad spectrum of physiological processes.
Kcnj
channel expression patterns generated in the present study were fitted with a deformable subdivision mesh atlas produced for the P14 rat brain. This co-registration, when combined with numerical quantification of expression strengths, allowed for semi-quantitative automated annotation of expression patterns as well as comparisons among and between
Kcnj
subfamilies. The expression patterns of
Kcnj
channel were also cross validated against previously published expression patterns of
Kcnj
channel genes.
This volume brings together a selection of contributions on the forms and functions of both contemporary and historical visual media. It aims to bring a range of perspectives from philosophical, ...cultural studies, and media studies on the materiality, semiotics, and aesthetics of images and other visual media forms into productive dialogue with each other.
Rapid progress in exploring the human and mouse genome has resulted in the generation of a multitude of mouse models to study gene functions in their biological context. However, effective screening ...methods that allow rapid noninvasive phenotyping of transgenic and knockout mice are still lacking. To identify murine models with bone alterations in vivo, we used flat-panel volume computed tomography (fpVCT) for high-resolution 3-D imaging and developed an algorithm with a computational intelligence system. First, we tested the accuracy and reliability of this approach by imaging discoidin domain receptor 2- (DDR2-) deficient mice, which display distinct skull abnormalities as shown by comparative landmark-based analysis. High-contrast fpVCT data of the skull with 200 microm isotropic resolution and 8-s scan time allowed segmentation and computation of significant shape features as well as visualization of morphological differences. The application of a trained artificial neuronal network to these datasets permitted a semi-automatic and highly accurate phenotype classification of DDR2-deficient compared to C57BL/6 wild-type mice. Even heterozygous DDR2 mice with only subtle phenotypic alterations were correctly determined by fpVCT imaging and identified as a new class. In addition, we successfully applied the algorithm to classify knockout mice lacking the DDR1 gene with no apparent skull deformities. Thus, this new method seems to be a potential tool to identify novel mouse phenotypes with skull changes from transgenic and knockout mice on the basis of random mutagenesis as well as from genetic models. However for this purpose, new neuronal networks have to be created and trained. In summary, the combination of fpVCT images with artificial neuronal networks provides a reliable, novel method for rapid, cost-effective, and noninvasive primary screening tool to detect skeletal phenotypes in mice.
Connecting fluorescence signals with anatomic structures enhances our ability to monitor biologic processes in mice. Here, we present a semiautomated approach to correlate two-dimensional (2D) ...noninvasive near-infrared fluorescence (NIRF) imaging with three-dimensional (3D), high-resolution, flat-panel volume computed tomography (fpVCT). We developed an algorithm to colocalize fluorescence signals of NIRF-labeled antibodies directed against matriptase and urokinase plasminogen activator receptor (uPAR) to orthotopic carcinomas in mice visualized by fpVCT. For this purpose, mice were anesthetized and fixed on a multimodality animal bed containing fiducial markers filled with iodine-containing contrast agent and fluorescent dye. After intravenous administration of contrast agent and Cy5.5-labeled antibodies, NIRF and fpVCT images were obtained, without repositioning the mice. Binding of Cy5.5-labeled matriptase-specific antibody to pancreatic tumors and Cy5.5-labeled uPAR-specific antibody to mammary carcinomas was assessed by time-domain NIRF imaging measuring the location of fluorescence intensity and its lifetime. In summary, we developed a novel 2D-3D registration technique for image fusion with NIRF imaging and fpVCT to provide complementary information in tumor models on the in vivo association of functional information with anatomic structures. The combination of fpVCT with NIRF imaging will now allow targeted and effective monitoring of preclinical tumor therapies.
Past, present and future of immunology in Mainz Waisman, Ari; Hövelmeyer, Nadine; Diefenbach, Andreas ...
Cellular immunology,
October 2016, 2016-10-00, 20161001, Volume:
308
Journal Article
PDF
