Mechanistic Studies of the Rho Helicase
All cellular organisms rely on transcriptional regulatory mechanisms to control the levels and timing of gene expression. Bacterial transcription is in part regulated by Rho, a RecA-family hexameric helicase that utilizes an ATP-dependent 5’→3’ translocation mechanism to terminate synthesis of specific RNA transcripts. Rho is initially recruited to pyrimidine-rich transcripts in an open-ring, loading-competent configuration, and subsequently traps the RNA within Rho’s central pore by transitioning to a closed-ring and catalytically active conformation. Using small-angle X-ray scattering and a novel fluorescence-based assay to monitor Rho’s conformational state in vitro, I discovered that bicyclomycin, a known small molecule inhibitor of Rho, acts by sterically blocking isomerization of Rho into its closed-ring form. Conversely, I also demonstrated that two distinct Rho effectors – pyrimidine-rich nucleic acids (as are found in favored RNA elements) and the transcription factor NusG – directly promote Rho ring closure. To better understand how NusG modulates Rho activity, I determined a crystal structure of closed-ring Rho in complex with the NusG C-terminal domain. This structure reveals that NusG engages the C-terminal face of Rho’s motor domains. Modeling of a Rho/NusG/RNAP complex based on the structure suggests that NusG may position Rho near the RNA exit channel of RNA polymerase to aid in the capture of non-ideal RNAs. Collectively, these findings delineate how a diverse set of ligands regulate bacterial transcription termination, and demonstrate how the conformational state of a hexameric helicase can be controlled by disparate classes of factors.