RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.
- MeSH
- bakteriální proteiny chemie metabolismus ultrastruktura MeSH
- DNA bakterií chemie metabolismus MeSH
- DNA řízené RNA-polymerasy chemie metabolismus ultrastruktura MeSH
- elektronová kryomikroskopie MeSH
- katalytická doména MeSH
- molekulární modely MeSH
- Mycobacterium smegmatis enzymologie MeSH
- nukleové kyseliny metabolismus MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396-His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp-His adduct mainly for binding and to a smaller extent for electron transfer.
- MeSH
- bilirubin metabolismus MeSH
- Hypocreales metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely * MeSH
- oxidace-redukce MeSH
- oxidoreduktasy působící na CH-CH vazby metabolismus MeSH
- transport elektronů fyziologie MeSH
- vazba proteinů fyziologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background: The rumen microbiota is one of the most complex consortia of anaerobes, involving archaea, bacteria, protozoa, fungi and phages. They are very effective at utilizing plant polysaccharides, especially cellulose and hemicelluloses. The most important hemicellulose decomposers are clustered with the genus Butyrivibrio. As the related species differ in their range of hydrolytic activities and substrate preferences, Butyrivibrio fibrisolvens was selected as one of the most effective isolates and thus suitable for proteomic studies on substrate comparisons in the extracellular fraction. The B. fibrisolvens genome is the biggest in the butyrivibria cluster and is focused on "environmental information processing" and "carbohydrate metabolism". Methods: The study of the effect of carbon source on B. fibrisolvens 3071 was based on cultures grown on four substrates: xylose, glucose, xylan, xylan with 25% glucose. The enzymatic activities were studied by spectrophotometric and zymogram methods. Proteomic study was based on genomics, 2D electrophoresis and nLC/MS (Bruker Daltonics) analysis. Results: Extracellular β-endoxylanase as well as xylan β-xylosidase activities were induced with xylan. The presence of the xylan polymer induced hemicellulolytic enzymes and increased the protein fraction in the interval from 40 to 80 kDa. 2D electrophoresis with nLC/MS analysis of extracellular B. fibrisolvens 3071 proteins found 14 diverse proteins with significantly different expression on the tested substrates. Conclusion: The comparison of four carbon sources resulted in the main significant changes in B. fibrisolvens proteome occurring outside the fibrolytic cluster of proteins. The affected proteins mainly belonged to the glycolysis and protein synthesis cluster.
- Publikační typ
- časopisecké články MeSH
The HelD is a helicase-like protein binding to Bacillus subtilis RNA polymerase (RNAP), stimulating transcription in an ATP-dependent manner. Here, our small angle X-ray scattering data bring the first insights into the HelD structure: HelD is compact in shape and undergoes a conformational change upon substrate analog binding. Furthermore, the HelD domain structure is delineated, and a partial model of HelD is presented. In addition, the unique N-terminal domain of HelD is characterized as essential for its transcription-related function but not for ATPase activity, DNA binding, or binding to RNAP. The study provides a topological basis for further studies of the role of HelD in transcription.
- MeSH
- Bacillus subtilis enzymologie MeSH
- bakteriální proteiny chemie metabolismus MeSH
- difrakce rentgenového záření MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.
- MeSH
- adenosinmonofosfát metabolismus MeSH
- bakteriální proteiny chemická syntéza chemie metabolismus MeSH
- jednovláknová DNA metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- Legionella pneumophila enzymologie MeSH
- molekulární modely MeSH
- nukleotidasy chemická syntéza chemie metabolismus MeSH
- proteiny - lokalizační signály fyziologie MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- RNA metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- sekvenční seřazení MeSH
- statická elektřina MeSH
- substrátová specifita MeSH
- teplota MeSH
- zinek chemie MeSH
- Publikační typ
- časopisecké články MeSH
The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.
- MeSH
- aminokyseliny metabolismus MeSH
- Aspergillus oryzae enzymologie metabolismus MeSH
- endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- katalytická doména fyziologie MeSH
- katalýza MeSH
- kinetika MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- substrátová specifita MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Pulmonary hypertension (PH), associated with imbalance in vasoactive mediators and massive remodeling of pulmonary vasculature, represents a serious health complication. Despite the progress in treatment, PH patients typically have poor prognoses with severely affected quality of life. Asymmetric dimethyl arginine (ADMA), endogenous inhibitor of endothelial nitric oxide synthase (eNOS), also represents one of the critical regulators of pulmonary vascular functions. The present study describes a novel mechanism of ADMA-induced dysfunction in human pulmonary endothelial and smooth muscle cells. The effect of ADMA was compared with well-established model of hypoxia-induced pulmonary vascular dysfunction. It was discovered for the first time that ADMA induced the activation of signal transducer and activator of transcription 3 (STAT3) and stabilization of hypoxia inducible factor 1α (HIF-1α) in both types of cells, associated with drastic alternations in normal cellular functions (e.g., nitric oxide production, cell proliferation/Ca(2+) concentration, production of pro-inflammatory mediators, and expression of eNOS, DDAH1, and ICAM-1). Additionally, ADMA significantly enhanced the hypoxia-mediated increase in the signaling cascades. In summary, increased ADMA may lead to manifestation of PH phenotype in human endothelial and smooth muscle cells via the STAT3/HIF-1α cascade. Therefore this signaling pathway represents the potential pathway for future clinical interventions in PH.
- MeSH
- amidohydrolasy metabolismus MeSH
- arginin analogy a deriváty farmakologie MeSH
- arteria pulmonalis účinky léků metabolismus patofyziologie MeSH
- endoteliální buňky účinky léků metabolismus MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus MeSH
- hypoxie buňky MeSH
- kultivované buňky MeSH
- lidé MeSH
- mediátory zánětu metabolismus MeSH
- mezibuněčná adhezivní molekula-1 metabolismus MeSH
- myocyty hladké svaloviny účinky léků metabolismus MeSH
- oxid dusnatý metabolismus MeSH
- plicní hypertenze etiologie metabolismus patofyziologie MeSH
- proliferace buněk účinky léků MeSH
- signální transdukce účinky léků MeSH
- svaly hladké cévní účinky léků metabolismus patofyziologie MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- transkripční faktor STAT3 metabolismus MeSH
- vápník metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
