Structural basis of RfaH-mediated transcription-translation coupling.

The NusG paralog RfaH mediates bacterial transcription-translation coupling on genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here we report cryo-EM structures of transcription-translation complexes (TTCs) containing RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP, and with the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs, due to the more restricted flexibility of the RfaH interdomain linker. The results further show that the structural organization of RfaH-coupled TTCs in the "loading state," in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the "loaded state," in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription-translation coupling. One sentence summary: Cryo-EM reveals the structural basis of transcription-translation coupling by RfaH.