Structure of the human Mediator-RNA polymerase II pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation at eukaryotic genes1–3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1–6. Here we prepare a recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and determine the structure of the complex by cryo-EM. Mediator uses its head module to contact the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7–9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the linker to the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate CTD phosphorylation

Development of a versatile laboratory experiment to teach the metabolic transformation of hydrolysis

In this paper we describe an easy, reliable, versatile and inexpensive laboratory experiment to teach the metabolic transformation of hydrolysis to Pharmacy students. The experiment does not require the sacrifice of any experimental animal, or any work with organs or tissues, and so can be implemented in a typical university chemistry laboratory. We used acetylsalicylic acid (ASA), hexyl salicylate (HS) and two enzymes, a lipase and an esterase. Since both ASS and HS liberate salicylic acid (SA) upon hydrolysis, students can evaluate the different enzymatic transformations by monitoring the amount of SA liberated. The learning outcomes are an enhanced student understanding of: (1) the process of hydrolysis; (2) the application of enzymatic transformations of molecules from food to xenobiotics; (3) the differences between the general specificity of substrate of both enzymes; (4) the concepts of the lipophilic pocket; (5) the catalytic triad and its regioselectivity in relation to the ester bond. A questionnaire was administered to participating students at three points in time: at the beginning of the module, after enzymatic hydrolysis was taught in class, and after the laboratory experiment. From an analysis of the questionnaire data we conclude that this practical helped Pharmacy students to understand these concepts

Conformational Transition Pathway in the Inhibitor Binding Process of Human Monoacylglycerol Lipase

Human monoacylglycerol lipase (MGL) catalyzes the hydrolysis of 2-arachidonoylglycerol to arachidonic and glycerol, which plays a pivotal role in the normal biological processes of brain. Co-crystal structure of the MGL in complex with its inhibitor, compound 1, shows that the helix α4 undergoes large-scale conformational changes in response to the compound 1 binding compared to the apo MGL. However, the detailed conformational transition pathway of the helix α4 in the inhibitor binding process of MGL has remained unclear. Here, conventional molecular dynamics (MD) and nudged elastic band (NEB) simulations were performed to explore the conformational transition pathway of the helix α4. Conventional MD simulations unveiled that the compound 1 induced the closed conformation of the active site of MGL, reduced the conformational flexibility of the helix α4, and elicited the large-scale conformational rearrangement of the helix α4, leading to the complete folding of the helix α4. Moreover, NEB simulations revealed that the conformational transition pathway of helix α4 underwent an almost 180° counter-clockwise rotation of the helix α4. Our computational results advance the structural and mechanistic understanding of the inhibitory mechanism

Passivation of recombination active PdSex centers in textured 001 oriented photoactive WSe2 films

Highly 001 textured tungsten diselenide WSe2 thin films are crystallized from X ray amorphous WSex films with Pd promotion in an H2Se atmosphere. During the crystallization process, the liquid promoter PdSex is driven to the grain boundaries due to the lateral growth of WSe2 platelets and their coalescence. The photoactivity of the WSe2 films is limited by the PdSex crystallites at the grain boundaries of WSe2 platelets. The presence of the PdSex crystallites leads to a metal like behavior of the conductivity versus temperature, whereas their etching leads to a semiconducting behavior of the WSe2 film. After selective etching of these crystallites, the more active WSe2 sites edges, cavities are exposed, enhancing the electron transfer to the electrolyte. A photocurrent density of 2 mA cm amp; 8722;2 at 0.35 V vs. reversible hydrogen electrode RHE is observed in sulfuric acid 0.5M H2SO4 using Fe2 Fe3 as redox pair under 1 sun Air Mass AM 1.5, 100mWcm amp; 8722;2 illuminatio

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2Å and in complex with phenylmethylsulfonyl fluoride at 1.8Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism

Discovery of BAY-985, a highly selective TBK1/IKK epsilon inhibitor.

The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKK epsilon are noncanonical members of the inhibitor of the nuclear factor kappa B (I kappa B) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKK epsilon inhibitor BAY-985. BAY-985 inhibits the cellular phosphorylation of interferon regulatory factor 3 and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2 but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model