Structure of the Bacterial Transcription Termination Factor Rho in Complex with the Regulatory Factor NusG

All eukaryotic and prokaryotic organisms utilize Spt5‐like proteins to regulate transcription. The bacterial Spt5 homolog NusG binds to RNA polymerase with its N‐terminal domain, and to transcription factors such as Rho with its C‐terminal domain. Rho is a hexameric helicase that utilizes energy from ATP hydrolysis to directionally translocate specific nascent transcripts through its central pore, which generates a force that disrupts transcription by RNA polymerase at distinct sites in bacterial genomes. For reasons that remain unclear, NusG is strictly required for a subset of transcripts at Rho‐dependent termination loci. In order to further elucidate the mechanism by which NusG aides Rho‐dependent transcription termination, we solved a 3.7 Å crystal structure of a complex of Rho, RNA, and the C‐terminal domain of NusG. The structure reveals that NusG binds to Rho in a fashion that orients Rho to translocate directly towards RNA polymerase, and positions the central pore of Rho near the RNA exit channel of the polymerase. We established both pulldown‐ and anisotropy‐based assays to track binding of NusG to Rho in vitro, and used the Rho/NusG complex structure to guide the engineering of point mutants that abrogate binding. We are using these mutants as tools to further probe the role of NusG in Rho‐dependent termination both in vitro and in vivo . Support or Funding Information This work was supported through funding from the G. Harold and Leila Y. Mathers Foundation and the NSF Graduate Research Fellowship Program.