Structural investigation of transcription reprogramming by E. coli stringent starvation protein A
EMSL Project ID
51039
Abstract
Stringent starvation protein A (SspA) is a global transcriptional regulator that associates with RNA polymerase (RNAP) and plays roles in the activation of the stringent response and pathogenesis in many gram-negative bacteria. SspA has been best characterized in E. coli where studies have shown SspA to be essential for acid-tolerance during stationary phase and late gene activation and lytic development of bacteriophage P1. Phage P1 encodes late promoter activator (Lpa) which is also required for lytic development. Lpa and SspA associate with the Ec RNAP ?70-containing holoenzyme to change its promoter specificity and redirect it to P1 late genes, facilitating late gene activation through an unknown mechanism, and permitting lytic development. Our goal is to further our understanding of the molecular mechanisms SspA uses to facilitate transcriptional reprogramming. We hypothesize that SspA regulates the transcription initiation step at acid resistance and P1 late gene promoters through direct interactions. This hypothesis will be tested through the completion of two specific aims. First, we propose to solve the structure of an E. coli RNAP-SspA-gadA acid stress promoter complex to determine how SspA associates with RNAP and regulates transcription activation at the gadA promoter. We have prepared grids of this complex that have not been screened yet. We anticipate that initial screening on the Talos Arctica followed by one or two days of data collection on the Titan Krios will be necessary to solve this structure. Our reconstructions from Aim 1 could provide a framework for rational inhibitor design to disrupt SspA function and therefore acid tolerance in enteric gram-negative bacterial pathogens. Our second aim is to elucidate the SspA/Lpa mediated mechanism of transcription activation at P1 late genes by solving the structure of a RNAP-SspA-Lpa-Ps late gene promoter complex. Structures from aim 2 would provide insight into how transcription factors interact with RNAP-SspA to regulate genes. So far, we have solved a preliminary structure of RNAP-SspA-Lpa-Ps complex using cryo-EM, revealing a novel mode of transcription factor binding to RNAP. Density for Lpa was not visible in our reconstructions; therefore, we decided to prepare grids of the complex after light treatment with glutaraldehyde. These grids have been screened on a Talos Arctica and a representative screening image can be found in the attached proposal. We anticipate two days of data collection on the Titan Krios will be necessary to image a sufficient number of particles.
Project Details
Start Date
2019-10-15
End Date
2020-07-15
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Travis B., K.M. Ramsey, S.M. Prezioso, T. Tallo, J.M. Wandzilak, A. Hsu, and M. Borgnia, et al. "Structural Basis for Virulence Activation of Francisella tularensis." Molecular Cell