The RNA polymerase found in Gram-positive bacteria is composed of multiple subunits, some of which exhibit interesting dynamic behaviors at various timescales. Nuclear magnetic resonance (NMR) is known as an unique technique capable of delivering insights into protein motions with atomic resolution. While analyzing the internal motion of structured proteins within the picosecond to nanosecond timescale relies on a well-established approach involving relaxation rate measurements and analysis [1], investigating the dynamics of intrinsically disordered proteins (IDPs) proves notably more challenging task for multiple reasons (complexity of motions, signal overlaps in NMR spectra, etc.). This study presents an exploration of the C-terminal domain of the delta subunit of RNA polymerase, which represents a disorder region posing particular investigative challenges. In order to obtain detailed information about significant, slower segmental motions occurring within the nanosecond timescale, a specialized NMR methodology called high-resolution relaxometry [2] was employed. This approach resulted in a collection of unprecedented amounts of data covering the relaxation of backbone amides under a broad range of magnetic fields spanning two orders of magnitude. In order to validate the analysis process, a novel NMR experiment was devised, enabling the measurement of relaxation rates at an exceptionally low magnetic field strength (0.33 T)[3] using a unique two-field NMR spectrometer [4]. This experiment successfully confirmed the applied protocol and analysis methods. Beyond its ability to study rapid picosecond-to-nanosecond motions, NMR enables the exploration of slower structural rearrangements through the analysis of relaxation dispersion experiments [5]. These experiments provide access to motions at the microsecond-to-millisecond timescale. Such motions are typically associated with important biological functions of biomolecules and they were detected also within domain 1.1 of the sigma subunit of the RNA polymerase. Analyzing the results of relaxation dispersion experiments revealed that the studied domain naturally inclines toward adopting a more disordered state, even at room temperature. By extending the study to multiple temperatures, the thermodynamic parameters associated with this transition were determined, and an increasing significance of the observed event with rising temperatures was predicted. Complementing the investigation was a functional characterization of domain 1.1 and results clearly proved the biological importance of the identified phenomena. References 1. Korzhnev, D.M., Billeter, M., Arseniev, A.S., & Orekhov, V.Y. (2001). NMR studies of Brownian tumbling and internal motions in proteins. Progress in Nuclear Magnetic Resonance Spectroscopy, 38 (3), 197-266. 2. Redfield A.G. (2003). Shuttling device for high-resolution measurements of relaxation and related phenomena in solution at low field, using a shared commercial 500 MHz NMR instrument. Magnetic Resonance in Chemistry, 41(10), 753-768 3. Jaseňáková Z., Zapletal V., Padrta P., Zachrdla M., Bolik-Coulon N., Marquardsen T., Tyburn J., Žídek L., Ferrage F., Kadeřávek P. (2020). Boosting the resolution of low-field 15N relaxation experiments on intrinsically disordered proteins with triple-resonance NMR. Journal of Biomolecular NMR, 74(2-3), 139-145 4. Cousin S.F., Charlier C., Kadeřávek P., Marquardsen T., Tyburn J.M., Bovier P.A., Ulzega S., Speck T., Wilhelm D., Engelke F., Maas W., Sakellariou D., Bodenhausen G., Pelupessy P., Ferrage F. (2016). High-resolution two-field nuclear magnetic resonance spectroscopy. Physical Chemistry Chemical Physics, 18(48), 33187–33194 5. Sekhar A., Kay L.E. (2013). NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. The Proceedings of the National Academy of Sciences, 110(32),12867-12874