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

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.

• Klíčová slova
• Bacillus, RNA polymerase, molecular modeling, nuclear magnetic resonance (NMR), protein structure, transcription initiation factor,
• 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
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• peptidové fragmenty MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny MeSH
• sigma faktor MeSH

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
• Názvy látek
• vnitřně neuspořádané proteiny 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
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• podjednotky proteinů MeSH

In bacteria, rapid changes in gene expression can be achieved by affecting the activity of RNA polymerase with small molecule effectors during transcription initiation. An important small molecule effector is the initiating nucleoside triphosphate (iNTP). At some promoters, an increasing iNTP concentration stimulates promoter activity, while a decreasing concentration has the opposite effect. Ribosomal RNA (rRNA) promoters from Gram-positive Bacillus subtilis are regulated by the concentration of their iNTP. Yet, the sequences of these promoters do not emulate the sequence characteristics of [iNTP]-regulated rRNA promoters of Gram-negative Escherichia coli. Here, we identified the 3'-promoter region, corresponding to the transcription bubble, as key for B. subtilis rRNA promoter regulation via the concentration of the iNTP. Within this region, the conserved -5T (3 bp downstream from the -10 hexamer) is required for this regulation. Moreover, we identified a second class of [iNTP]-regulated promoters in B. subtilis where the sequence determinants are not limited to the transcription bubble region. Overall, it seems that various sequence combinations can result in promoter regulation by [iNTP] in B. subtilis. Finally, this study demonstrates how the same type of regulation can be achieved with strikingly different promoter sequences in phylogenetically distant species.

• MeSH
• Bacillus subtilis genetika   metabolismus   MeSH
• DNA bakterií chemie   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• Escherichia coli enzymologie   MeSH
• genetická transkripce * MeSH
• geny rRNA MeSH
• kinetika MeSH
• nukleotidy metabolismus   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• nukleotidy MeSH

Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of (13)C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• bakteriální proteiny chemie   MeSH
• DNA řízené RNA-polymerasy chemie   MeSH
• izotopy uhlíku MeSH
• konformace proteinů MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• proteiny chemie   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy uhlíku MeSH
• proteiny MeSH

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.

• MeSH
• algoritmy MeSH
• deuterium MeSH
• Fourierova analýza MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• molekulární sekvence - údaje MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• proteiny chemie   MeSH
• repetitivní sekvence nukleových kyselin * MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deuterium MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• proteiny MeSH