Spectral density mapping represents the method of choice for investigations of molecular motions of intrinsically disordered proteins (IDPs). However, the current methodology has been developed for well-folded proteins. In order to find conditions for a reliable analysis of relaxation of IDPs, accuracy of the current reduced spectral density mapping protocols applied to IDPs was examined and new spectral density mapping methods employing cross-correlated relaxation rates have been designed. Various sources of possible systematic errors were analyzed theoretically and the presented approaches were tested on a partially disordered protein, delta subunit of bacterial RNA polymerase. Results showed that the proposed protocols provide unbiased description of molecular motions of IDPs and allow to separate slow exchange from fast dynamics.

• MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• sbalování proteinů MeSH
• vnitřně neuspořádané proteiny analýza   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.

• MeSH
• Bacillus subtilis metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• molekulární modely * MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sigma faktor chemie   genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• strukturní homologie proteinů MeSH
• Thermotoga maritima enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

• MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• druhová specificita MeSH
• genetická transkripce * MeSH
• genetické techniky MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií * MeSH
• sekvence nukleotidů MeSH
• sigma faktor genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

σ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ factors occupies the primary channel of RNAP and also prevents binding of the σ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σA exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α helices, while the second α helix and β strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σA and its shortened version lacking domain 1.1 ( σA_Δ1.1 ). The results revealed that while full length σA increases transcription activity of RNAP with increasing temperature, transcription with σA_Δ1.1 remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.

• MeSH
• amidy metabolismus   MeSH
• Bacillus subtilis * enzymologie   MeSH
• DNA řízené RNA-polymerasy * chemie   metabolismus   MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• protony MeSH
• rozbalení proteinů * MeSH
• sigma faktor * chemie   metabolismus   MeSH
• teplota * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well-folded N-terminal domain was determined based on inter-proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C-terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C-terminal domain. Various (15) N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C-terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C-terminal tail with the N-terminal domain and with itself were identified. A propensity of the C-terminal domain to form β-type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well-balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C-terminal domain. The results showed that the δ subunit consists of a well-ordered N-terminal domain and a flexible C-terminal domain that exhibits a complex hierarchy of partial ordering.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny biosyntéza   chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Description of protein dynamics is known to be essential in understanding their function. Studies based on a well established [Formula: see text] NMR relaxation methodology have been applied to a large number of systems. However, the low dispersion of [Formula: see text] chemical shifts very often observed within intrinsically disordered proteins complicates utilization of standard 2D HN correlated spectra because a limited number of amino acids can be characterized. Here we present a suite of triple resonance HNCO-type NMR experiments for measurements of five [Formula: see text] relaxation parameters ([Formula: see text], [Formula: see text], NOE, cross-correlated relaxation rates [Formula: see text] and [Formula: see text]) in doubly [Formula: see text],[Formula: see text]-labeled proteins. We show that the third spectral dimension combined with non-uniform sampling provides relaxation rates for almost all residues of a protein with extremely poor chemical shift dispersion, the C terminal domain of [Formula: see text]-subunit of RNA polymerase from Bacillus subtilis. Comparison with data obtained using a sample labeled by [Formula: see text] only showed that the presence of [Formula: see text] has a negligible effect on [Formula: see text], [Formula: see text], and on the cross-relaxation rate (calculated from NOE and [Formula: see text]), and that these relaxation rates can be used to calculate accurate spectral density values. Partially [Formula: see text]-labeled sample was used to test if the observed increase of [Formula: see text] [Formula: see text] in the presence of [Formula: see text] corresponds to the [Formula: see text] dipole-dipole interactions in the [Formula: see text],[Formula: see text]-labeled sample.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• vodík MeSH
• Publikační typ
• časopisecké články MeSH

The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni(2+) ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů * MeSH
• krystalografie rentgenová metody   MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

RNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the δ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of δ from Bacillus subtilis. We demonstrate that δ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that δ is essential for cell survival when facing a competing strain in a changing environment. Hence, although δ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis.

• MeSH
• Bacillus subtilis enzymologie   genetika   fyziologie   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• exprese genu MeSH
• fyziologická adaptace MeSH
• genetická transkripce MeSH
• genový knockout MeSH
• mikrobiální viabilita MeSH
• podjednotky proteinů MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u bakterií genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Bacterial RNA polymerase (RNAP) is an essential multisubunit protein complex required for gene expression. Here, we characterize YvgS (HelD) from Bacillus subtilis, a novel binding partner of RNAP. We show that HelD interacts with RNAP-core between the secondary channel of RNAP and the alpha subunits. Importantly, we demonstrate that HelD stimulates transcription in an ATP-dependent manner by enhancing transcriptional cycling and elongation. We demonstrate that the stimulatory effect of HelD can be amplified by a small subunit of RNAP, delta. In vivo, HelD is not essential but it is required for timely adaptations of the cell to changing environment. In summary, this study establishes HelD as a valid component of the bacterial transcription machinery.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• Bacillus subtilis enzymologie   genetika   MeSH
• bakteriální proteiny izolace a purifikace   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   izolace a purifikace   metabolismus   MeSH
• DNA metabolismus   MeSH
• elongace genetické transkripce MeSH
• fenotyp MeSH
• genetická transkripce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i-iii. Lipophosphonoxins displayed significant antibacterial properties against a panel of Gram-positive species, including multiresistant strains. The minimum inhibitory concentration (MIC) values of the best inhibitors were in the 1-12 μg/mL range, while their cytotoxic concentrations against human cell lines were significantly above this range. The modular nature of this artificial scaffold offers a large number of possibilities for further modifications/exploitation of these compounds.

• MeSH
• antibakteriální látky chemická syntéza   chemie   farmakologie   MeSH
• apoptóza účinky léků   MeSH
• erytroidní prekurzorové buňky cytologie   účinky léků   MeSH
• fetální krev MeSH
• grampozitivní bakterie účinky léků   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kultivované buňky MeSH
• kyseliny fosforité chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence MeSH
• nukleosidy chemická syntéza   chemie   farmakologie   MeSH
• stereoizomerie MeSH
• viabilita buněk účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH