Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, are able to dissociate rifampicin-stalled RNAPs from DNA and provide rifampicin resistance. This is achieved by the ability of HelR proteins to dissociate rifampicin from RNAP. The HelR-mediated mechanism of rifampicin resistance is discussed here, and the roles of HelD/HelR in the transcriptional cycle are outlined. Moreover, the possibility that the structurally similar HelD proteins from low G+C Firmicutes may be also involved in rifampicin resistance is explored. Finally, the discovery of the involvement of HelR in rifampicin resistance provides a blueprint for analogous studies to reveal novel mechanisms of bacterial antibiotic resistance.

• Klíčová slova
• HelD/HelR, RNA polymerase, antibiotics, bacteria, resistance, rifampicin,
• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria * genetika   metabolismus   MeSH
• bakteriální léková rezistence MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• DNA MeSH
• rifampin * farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• rifampin * MeSH

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.

• MeSH
• amidy MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• simulace molekulární dynamiky MeSH
• vnitřně neuspořádané proteiny * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amidy MeSH
• DNA řízené RNA-polymerasy MeSH
• vnitřně neuspořádané proteiny * MeSH

Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• termodynamika MeSH
• vnitřně neuspořádané proteiny * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• vnitřně neuspořádané proteiny * MeSH

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone 15 N amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of δ subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

• Klíčová slova
• Dynamics, High-resolution relaxometry, Intrinsically disordered proteins, Non-uniform sampling, Nuclear magnetic resonance, Relaxation,
• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• rekombinantní proteiny chemie   MeSH
• vnitřně neuspořádané proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• rekombinantní proteiny MeSH
• vnitřně neuspořádané proteiny MeSH