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Mechanistic Studies of the Rho Helicase

  • Author(s): Lawson, Michael Robert
  • Advisor(s): Berger, James
  • Rio, Donald
  • et al.

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.

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