Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro.

In the conventional model of the Rho-dependent transcription termination, the terminator Rho binds to the rut (Rho utilization) site and translocates along the nascent RNA prior to making possible interactions with the elongating RNA polymerase (RNAP). Even though the interaction between Rho and isolated RNAs was studied in great detail, the same has never been shown with the nascent RNA emerging from the transcription elongation complex (EC). Direct demonstration and requirement of the Rho-nascent RNA binding become even more important because of the recently proposed alternative model where Rho loads onto the RNAP prior to the formation of the nascent RNA. Here, we have measured the direct association of Rho in vitro with the free RNAP, RNAP-promoter binary complex and stalled ECs with varied length of RNA. We observed the association of Rho only with the ECs having the rut-site-containing long nascent RNA. This association was significantly reduced when either a Rho mutant, Y80C, defective for RNA binding or an antisense oligo to the rut site was used or when the rut site was eliminated by RNase digestion or replacement with a random RNA sequence. The presence of EC-bound NusG, the binding partner of Rho, did not facilitate this association. RNase footprinting of the Rho-EC complex revealed a clear Rho-mediated protection of the rut sites on the nascent RNA. We concluded that the nascent RNA loading of Rho and its interaction with the rut site are mandatory and prerequisites for its recruitment to the EC under in vitro experimental conditions.