The unstructured C-terminal domain of delta subunit of bacterial RNA Polymerase is 90 aa long and highly charged. The charge distribution of this domain is distinct, with a conserved stretch of 9 residues (96−104) containing 7 positive charges followed by the rest of the domain with 51 acidic residues (K-D/E motif). A previous study demonstrated that the two parts of the motif transiently interact, and this affects the spatiotemporal properties of this domain. From the biological point of view, δ increases cell fitness and virulence of pathogens and was previously proposed to function as a nucleic acid mimic and affect RNAP− nucleic acid interactions.
Lukáš Žídek Research Group
Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in-vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.
Kuban, V., Srb, P., Stegnerova, H., Padrta, P., Zachrdla, M., Jasenakova, Z., Sanderova, H., Vitovska, D., Krasny, L..,Koval, T., Dohnalek, J., Ziemska-Legiecka, J., Grynberg, M., Jarnot, P., Gruca, A., Jensen, M.R., Blackledge, M., and Zidek, L.: Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase, J. Am. Chem. Soc. 2019, 141, 16817-16828, DOI:10.1021/jacs.9b07837