The transcription of genetic information from DNA into RNA is carried out by the multi-subunit enzyme RNA polymerase (RNAP) and is a fundamental process in cellular organisms. RNAP requires an entourage of transcription factors to modulate transcription. The multi-domain NusA protein is an essential transcription factor that is confined to bacteria, regulating transcription rates, termination efficiency and pausing. Transcriptional pausing is required for coupling transcription and translation, RNA folding, allowing recruitment of regulatory proteins, and is the first step in termination. Structurally, NusA can be divided into two domains, the amino-terminal domain (NTD) that binds directly to RNAP and is responsible for pausing activity, and the carboxy-terminal domain (CTD) that contains multiple RNA interacting motifs. We previously solved a low-resolution structure of NusA in complex with RNAP, showing that the NusA NTD binds RNAP at the ß-flap, a region directly adjacent the RNA exit channel. We have now determined high-resolution structures of NusA and RNAP ß-flap. We have identified that the ß-flap tip region is highly flexible and determined the interaction site for NusA. NusA also appears to undergo significant conformational change upon binding to the ß-flap. Preliminary modelling suggested that a large basic patch of the NusA NTD is oriented towards the RNA exit channel on RNAP. Transcriptional pausing involves the formation of a short RNA hairpin and all the basic amino acids on NusA NTD that face the RNA exit channel were mutagenized. All of these basic residues play a role in stabilising the transcription pause hairpin, but two particular amino acids are absolutely required and have a non-redundant function. We propose a model for transcription pausing where NusA bound to the ß-flap of RNAP is able to stabilise transcriptional pausing via an interaction with the phosphodiester backbone of the A-form RNA duplex in the pause hairpin.