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Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An 'expander' element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A 'connector' element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase 'core' and 'shelf' modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core-shelf interface that includes a 'composite' active site for RNA chain initiation, elongation, proofreading and termination., Eukaryotic genes are transcribed by Pol I, II and III (ref. 1), which synthesize ribosomal, messenger and transfer RNA, respectively. Pol I synthesizes about 60% of cellular RNA by transcribing [...]

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 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the 'B-linker' that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the 'B-reader that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation., Transcription of eukaryotic protein-coding genes begins with formation of the pre-initiation complex (PIC) on promoter DNA. The PIC contains Pol II and the general transcription factors, including B and the [...]

Author(s): Liudmila Andreeva [1, 2]; Bjrn Hiller [1, 2]; Dirk Kostrewa [1, 2]; Charlotte Lssig [1, 2]; Carina C. de Oliveira Mann [1, 2]; David Jan Drexler [1, 2]; Andreas [...]

Research has been conducted on Aspergillus fumigatus. The pH activity of this bacterium and the consensus phytases have been engineered via the use of chemical modification and site-directed mutagenesis and the details are presented.

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however. functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH4+ or NH3) of the transported species. We have solved the structure of wild-type AmtB from Escherichla coil in two crystal forms at 1.8- and 2.1-Å resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH4+ is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH3. The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transport- ers/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner. [ABSTRACT FROM AUTHOR]

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte transmigration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 Å resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short tinker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by transinteractions of their N-terminal domains with JAM dimers from an opposite cell surface. [ABSTRACT FROM AUTHOR]

During gene transcription, the RNA polymerase (Pol) active center can catalyze RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III but very weak for Pol II. The reason for this difference is unclear because the active centers of the polymerases are virtually identical. Here we show that Pol II gains strong cleavage activity when the C-terminal zinc ribbon domain (C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III subunit C11. X-ray analysis shows that the C-ribbon has detached from its site on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon transiently inserts into the Pol II pore to complement the active center. This mechanism is also used by transcription factor IIS, a factor that can bind Pol II and induce strong RNA cleavage. Together with published data, our results indicate that Pol I and Pol III contain catalytic C-ribbons that complement the active center, whereas Pol II contains a non-catalytic C-ribbon that is immobilized on the enzyme surface. Evolution of the Pol II system may have rendered mRNA transcript cleavage controllable by the dissociable factor transcription factor IIS to enable promoter-proximal gene regulation and elaborate 3ߧ-processing and transcription termination. [ABSTRACT FROM AUTHOR]

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these sub-families is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases. [ABSTRACT FROM AUTHOR]

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, the Saccharomyces cerevisiae enzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The unique beta-hydroxyacyl-ACP dehydratase in Plasmodium falciparum, PfFabZ, is involved in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxy fatty acids linked to acyl carrier protein. The structure was solved by single anomalous dispersion (SAD) phasing using a quick-soaking experiment with potassium iodide and refined to a resolution of 2.1 A. The crystal structure represents the first structure of a Plasmodium beta-hydroxyacyl-ACP dehydratase with broad substrate specificity. The asymmetric unit contains a hexamer that appears as a trimer of dimers. Each dimer shows the known "hot dog" fold that has been observed in only a few other protein structures. Each of the two independent active sites in the dimer is formed by equal contributions from both subunits. The active site is mainly hydrophobic and looks like an L-shaped tunnel. The catalytically important amino acids His 133 and Glu 147' (from the other subunit), together with His98', form the only hydrophilic site in this tunnel. The inner end of the active site tunnel is closed by the phenyl ring of Phe 169, which is located in a flexible, partly visible loop. In order to explain the acceptance of substrates longer than ~C-7, the phenyl ring must move away to open the tunnel. The present structure supports an enzymatic mechanism consisting of an elimination reaction catalyzed by His 133 and Glu147'. 3-decynoyl-N-acetylcysteamine, an inhibitor known to interact with the E. coli dehydratase/isomerase, turned out to interact covalently with PfFabZ. A first model of PfFabZ with this potent inhibitor is presented.