Pausing during transcription elongation is a fundamental activity in all kingdoms of life. NusA is an essential RNA polymerase (RNAP) binding transcription factor that is unique to bacteria, and therefore, provides a compelling target for development of novel antibiotic leads. Although it has been known that NusA is important in controlling transcription, the mechanism by which it is able to do so has remained elusive for over 40 years.^{1, 2} This lack of understanding has limited efforts to target the NusA-RNAP interaction for drug development.

Here, we present the high-resolution NMR structure of the N-terminal domain of NusA (NusA-NTD) from the Gram positive model B. subtilis, as well as its RNAP-binding domain known as the β-flap. The interaction site of both proteins was mapped using using ¹⁵N chemical shift titration experiments. Our results show that the β-flap domain binds NusA via a short and highly dynamic helix. In contrast, NusA-NTD undergoes a conformational rearrangement upon binding. In order to differentiate between the binding site and the structural rearrangement, we performed extensive mutagenesis studies coupled with in vitro assays. These results showed that the RNAP-binding site of NusA is limited to a β-sheet region while the interaction causes allosteric rearrangement of a distal helical bundle. Further in vitro studies revealed a previously unknown segment of this helical bundle that is critical for the interaction of NusA with the emerging RNA transcript.

These results provide a mechanism by which the essential transcription factor NusA regulates transcriptional pausing, and provides an opportunity for targeting this interaction for drug design.