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.

Institut de Biologie Structurale Université Grenoble Alpes CEA CNRS 71 avenue des Martyrs Grenoble 38044 France

Institute of Biochemistry and Biophysics PAS Pawinskiego 5A Warsaw 02 106 Poland

Institute of Informatics Silesian University of Technology Akademicka 16 Gliwice 44 100 Poland

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences v v i Flemingovo nám 2 Prague 6 16610 Czech Republic

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology of the Czech Academy of Sciences v v i Vídenská 1083 Prague 4 14220 Czech Republic

Laboratory of Structure and Function of Biomolecules Institute of Biotechnology of the Czech Academy of Sciences v v i Biocev Prumyslová 595 Vestec 25250 Czech Republic

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