Integrative structural analysis of the vRNAP-coupled Coliphage N4 ejectosome

About This Grant

ABSTRACT Bacteriophage N4, the model virus of the newly established and understudied Schitoviridae family, employs a unique mechanism to translocate its genome into Gram-negative bacterial hosts. This process is mediated by gp50, a massive 380 kDa virion-associated RNA polymerase (vRNAP). N4 vRNAP's role in initiating transcription immediately after infection is well studied; however, its role in forming a specialized membrane- spanning complex termed the "ejectosome" is less understood. The transcriptional activity of this protein is carried out by the middle 1100 residues; however, domains of equal or greater length surround the transcriptional core on both the N and C termini, and these domains remain largely uncharacterized structurally and functionally. I have recently determined structures of the gp50 RNAP and C-terminal domains (currently under review), but the N-terminal domain and the complete ejectosome complex remain unresolved. This proposal addresses these knowledge gaps by leveraging state-of-the-art cryogenic electron microscopy (Cryo- EM) and electron tomography (Cryo-ET) approaches. I hypothesize that multiple copies of vRNAP (gp50) are structurally integrated into a membrane-spanning channel formed by N4 ejection proteins gp51 and gp52, with the N-terminal domain (NTD) of vRNAP contributing to the channel structure while the RNAP and C-terminal domains remain tethered to facilitate DNA translocation. In Aim 1, I will reconstitute the complete ejectosome complex in vitro from purified components (gp50-NTD, gp51, and gp52) and determine its high-resolution structure using Cryo-EM single particle analysis, revealing the structure of the N-terminal domain and its integration into the membrane-spanning channel. In Aim 2, I will use in situ Cryo-ET to visualize the N4 ejectosome in its pre- and post-ejection states. By analyzing mature virions and N4-infected bacterial minicells, I will capture the internal organization of vRNAP and associated ejection proteins before and after translocation. These experiments will provide insight into the structural transitions required for ejectosome assembly and function during early infection. Together, these studies will advance our understanding of the molecular mechanisms underlying genome ejection in N4 and related phages. Understanding the structural basis of genome ejection in this therapeutically relevant phage family has implications for phage therapy development, rational engineering of phage-based delivery systems, and microbiome dynamics. This work will provide me with comprehensive training in integrative structural biology approaches, including advanced Cryo- EM and Cryo-ET techniques, preparing me for an independent research career.